Stable, protracted glp-1/glucagon receptor co-agonists for medical use

ABSTRACT

The invention relates to novel stable and protracted GLP-1/glucagon receptor co-agonists, to the use of said peptides in therapy, to methods of treatment comprising administration of said peptides to patients, and to the use of said peptides in the manufacture of medicaments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/179,316, filed Jun. 10, 2016, which is a continuation of U.S.application Ser. No. 14/836,192 filed Aug. 26, 2015 (now U.S. Pat. No.9,474,790), which is a continuation of International ApplicationPCT/EP2014/058084 (WO 2014/170496), filed Apr. 22, 2014, which claimspriority to European Patent Application 13164272.0, filed Apr. 18, 2013and European Patent Application 13196656.6, filed Dec. 11, 2013; thisapplication claims priority under 35 U.S.C. §119 to U.S. ProvisionalApplication 61/814,969; filed Apr. 23, 2013; the contents of which areincorporated herein by reference.

TECHNICAL FIELD

The invention relates to novel stable and protracted GLP-1/glucagonreceptor co-agonists, to their use in therapy, to methods of treatmentcomprising administration hereof to patients, and to the use hereof inthe manufacture of medicaments.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 10, 2016, isnamed 8676US03SeqList_ST25.txt and is 6,919 bytes in size.

BACKGROUND

The increase in obesity incidence has reached epidemic proportions inthe western world and more recently also in developing countries.Obesity is associated with significant co-morbidities such ascardiovascular diseases and Type 2 diabetes. Presently, the onlytreatment that eliminates obesity with high efficacy is bariatricsurgery, but this treatment is costly and risky. Pharmacologicalintervention is generally less efficacious and associated with sideeffects. There is therefore an obvious need for more efficaciouspharmacological intervention with fewer side effects and convenientadministration.

Numerous gastro-intestinal peptide hormones are allegedly involved inthe regulation of food intake, being either anorexigenic (e.g. CCK,GLP-1, PYY, secretin) or orexigenic (e.g. ghrelin) [Moran T H: Gutpeptides in the control of food intake; Int. J. Obes. (Lond). 2009 33S7-10]. Recently, oxyntomodulin, a product from the proglucagon gene inintestinal L-cells was shown to induce satiety and reduce body weight inboth rodents and humans [Cohen M A et al: Oxyntomodulin suppressesappetite and reduces food intake in humans; J. Clin. Endocrinol. Metab.2003 88 4696-4701; Dakin C L et al: Oxyntomodulin inhibits food intakein the rat; Endocrinology 2001 142 4244-4250]. Oxyntomodulin is a dualagonist activating both GLP-1 and glucagon receptors, albeit withreduced potency compared to GLP-1 and glucagon, respectively. Theanorexigenic effect of oxyntomodulin was previously speculated to bemediated by the GLP-1 receptor, although numerous older studiesindicated the involvement of pancreatic glucagon in the control ofbodyweight. Two recent papers allegedly show glucagon as an attractivetarget and demonstrated the power of simultaneous GLP-1/glucagonreceptor-targeting by constructing dual agonists and comparing theweight lowering effect in knock-out models [Pocai et al; Glucagon-LikePeptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice;Diabetes, 2009, 58, 2258-2266; Day et al; A new GLP-1 co-agonisteliminates obesity in rodents; Nat. Chem. Biol., 2009, 5, 749-757].

One physiological effect of glucagon is to increase blood glucose levelsin hypoglycaemic conditions by stimulating glycogenolysis andgluconeogenesis. However, the acute effect of glucagon on blood glucoselevels seems to be modest when glucagon is infused at near-physiologicallevels [Sherwin R S et al: Hyperglucagonemia and blood glucoseregulation in normal, obese and diabetic subjects; N. Engl. J. Med.1976, 294, 455-461]. Glucagon receptor activation has also been shown toincrease energy expenditure and decrease food intake in both rodents andhumans [Habegger K M et al: The metabolic actions of glucagon revisited;Nat. Rev. Endocrinol. 2010 6 689-697] and these effects are robust andsustained in rodents. The risk of increased blood glucose levels due toglucagon agonism may be counter-acted by appropriate levels of GLP-1agonism. A GLP-1/glucR co-agonist with a balanced effect on the tworeceptors may give rise to an improved weight loss compared to a pureGLP-1 agonist without compromising the glucose tolerance. However, thereare several obstacles in developing such a co-agonist to apharmaceutical product, relating to half-life, stability, solubility andreceptor activity. For example, if glucagon is used as a starting pointfor such a co-agonist, the GLP-1 receptor activity needs to beestablished without destroying the activity at the glucagon receptor.Furthermore, since glucagon is inherently insoluble at neutral pH, it ischemically and physically unstable and its half-life in vivo is only afew minutes.

Several patent applications disclosing different GLP-1/glucagon receptorco-agonists are known in the art, e.g. WO 2008/101017, WO 2010/070255,WO 2012/150503, and WO 2012/169798.

In summary, there are several obstacles in developing such a co-agonistinto pharmaceutical products, in particular:

i) Receptor activity—The glucagon and GLP-1 receptor potency and/orbinding affinity ratio of the co-agonist should be balanced in order tofavour a robust reduction in body weight, without compromising glucosebalance;

ii) A protracted profile of action, i.e. an in vivo half-life thatallows dosing for example once a day or once a week; and

iii) Acceptable solubility, chemical and physical stability.

When glucagon is used as a starting point for such a co-agonist, theGLP-1 receptor activity and/or affinity needs to be established withoutdestroying the glucagon activity. Native glucagon is inherentlyinsoluble at neutral pH, it is chemically and physically unstable andits half-life in vivo is only a few minutes.

SUMMARY OF THE INVENTION

The invention relates to novel stable and protracted GLP-1/glucagonreceptor co-agonists (also referred to as “peptides” or “derivatives”herein, in particular referred to as “glucagon derivatives” herein), tothe use of said peptides in therapy, to methods of treatment comprisingadministration of said peptides to patients, and to the use of saidpeptides in the manufacture of medicaments for use in medicine,including the treatment of diabetes, obesity and related diseases andconditions.

In a first embodiment, the invention relates to a glucagon derivativecomprising the amino acid sequence of Formula I (corresponding to SEQ IDNO:4 and SEQ ID NO:5):

His-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₁₇-X₁₈-Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀  [I],

wherein,

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu, Ile or Val;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, Ala or Lys;

X₂₀ represents Gln, Arg, Glu, Aib or Lys;

X₂₁ represents Asp, Glu, Ser, or Lys;

X₂₄ represents Gln, Ala, Arg, Glu, Aib or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys;

X₂₉ represents Thr, Gly, Ser, Gln, Ala, Glu or Lys;

X₃₀ represents Lys, or X₃₀ is absent;

which amino acid sequence comprises a lysine residue at one or more ofpositions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30; and

wherein said glucagon derivative comprises a substituent comprising alipophilic moiety and at least three negatively charged moieties,wherein one of said negatively charged moieties is distal of saidlipophilic moiety, and wherein said substituent is attached at theepsilon position of a lysine residue in one of the amino acid positions16, 17, 18, 20, 21, 24, 28, 29, or 30; and wherein said glucagonderivative is a C-terminal amide; or a pharmaceutically acceptable saltor prodrug thereof.

In one embodiment the invention relates to a pharmaceutical compositioncomprising a glucagon derivative according the invention and optionallyone or more pharmaceutically acceptable excipients.

In one embodiment the invention relates to an intermediate product inthe form of a glucagon peptide comprising a C-terminal amide and any oneof the modifications a) to ppp) as defined herein as compared toglucagon (SEQ ID NO: 1), or a pharmaceutically acceptable salt, amide,or ester thereof.

In one embodiment the invention relates to a glucagon derivative of theinvention, optionally in combination with one or more additionaltherapeutically active compounds, for use in medicine.

The invention may solve further problems that will be apparent from thedisclosure of the exemplary embodiments.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the ThT fibrillation assay results described in Example76.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel glucagon derivatives which areGLP-1/glucagon receptor co-agonists with a novel amino acid mutation,which in combination with other mutations and a substituent withnegatively charged moieties, provide glucagon derivatives that activateboth the GLP-1 and glucagon receptors. The inventors have found that,for example, the introduction of a leucine in position 10, in analogueswith a substituent with at least three negative charges in addition toother substitutions gives rise to GLP-1/glucagon receptor co-agonistswith improved physical stability, i.e. the analogues show none ordelayed fibrillation in the assay used to assess physical stability andthe recovery of the peptides were in general improved. Furthermore, theinventors have surprisingly found that the glucagon derivatives of theinvention tend to reduce glucagon receptor binding and at the same timeoften improve the GLP-1 receptor binding. Therefore, the novelmutation(s) can be used as a tool for adjusting the ratio betweenglucagon and GLP-1 affinity which is pivotal for obtaining the desiredeffect on body weight and maintain blood glucose levels.

The inventors have found that the compounds of the invention haveadequate aqueous solubility at neutral pH or slightly basic pH and withimproved chemical stability i.e. the chemical degradation of theanalogues are reduced. The inventors have found that the compounds ofthe invention have improved pharmacokinetic properties, i.e. they haveprolonged half-life in vivo. Furthermore, the compounds of the inventioninduce a significant reduction in body weight after s.c. administration.

The glucagon derivatives of the invention may in particular becharacterised as a glucagon derivative comprising the amino acidsequence of Formula I (corresponding to SEQ ID NO:4 and SEQ ID NO:5):

His-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₁₇-X₁₈-Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀  [I]

wherein

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu, Ile or Val;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, Ala or Lys;

X₂₀ represents Gln, Arg, Glu, Aib or Lys;

X₂₁ represents Asp, Glu, Ser or Lys;

X₂₄ represents Gln, Ala, Arg, Glu, Aib or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys;

X₂₉ represents Thr, Gly, Ser, Gln, Ala, Glu or Lys;

X₃₀ represents Lys, or X₃₀ is absent;

which amino acid sequence comprises a lysine residue at one or more ofpositions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30; and whereinsaid glucagon derivative comprises a substituent comprising a lipophilicmoiety and at least three negatively charged moieties, wherein one ofsaid negatively charged moieties is distal of a lipophilic moiety, isattached at the epsilon position of a lysine residue in one of the aminoacid positions 16, 17, 18, 20, 21, 24, 28, 29, or 30; and wherein saidglucagon derivative is a C-terminal amide, or a pharmaceuticallyacceptable salt or prodrug thereof.

In one embodiment the glucagon derivative comprises the amino acidsequence of Formula I (corresponding to SEQ ID NO:2 and SEQ ID NO:3):

His-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₁₇-X₈-Ala-X₂₀-X₂₁₋Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀  [I]

whereinX₂ represents Aib, Acb or Acpr;X₃ represents Gln or His;X₁₀ represents Leu, Ile or Val;X₁₂ represents Lys or Arg;X₁₅ represents Asp or Glu;X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys;X₁₇ represents Arg or Lys;X₁₈ represents Arg, Ala or Lys;X₂₀ represents Gln, Arg, Glu, Aib or Lys;X₂₁ represents Asp, Glu or Lys;X₂₄ represents Gln, Ala, Arg, Glu, Aib or Lys;X₂₇ represents Met, Leu or Val;X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys;X₂₉ represents Thr, Gly, Ser, Gln, Ala, Glu or Lys;X₃₀ represents Lys, or X₃₀ is absent;which amino acid sequence comprises a lysine residue at one or more ofpositions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30; and whereinsaid glucagon derivative comprises a substituent comprising a lipophilicmoiety and at least three negatively charged moieties, wherein one ofsaid negatively charged moieties is distal of a lipophilic moiety, isattached at the epsilon position of a lysine residue in one of the aminoacid positions 16, 17, 18, 20, 21, 24, 28, 29, or 30; and wherein saidglucagon derivative is a C-terminal amide, or a pharmaceuticallyacceptable salt or prodrug thereof.

In one embodiment the invention relates to glucagon derivative peptides,wherein said glucagon derivative peptides are GLP-1 and glucagonreceptor co-agonists.

Glucagon Peptide

The peptide of the glucagon derivatives of the invention may bedescribed by reference to i) the number of the amino acid residues inhuman glucagon (SEQ ID NO: 1) which corresponds to the amino acidresidue which is modified (i.e. the corresponding position in glucagon(SEQ ID NO: 1)), and to ii) the actual modification. As regards positionnumbering in glucagon compounds: for the present purposes any amino acidsubstitution, deletion, and/or addition is indicated relative to thesequences of native human glucagon (1-29) (SEQ ID NO:1). Human glucagonamino acids positions 1-29 are herein to be the same as amino acidpositions X₁ to X₂₉. The human glucagon (1-29) sequence isHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-GIn-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO:1). Glucagon(1-30)means human glucagon with an extension of one amino acid in theC-terminal, glucagon(1-31) means human glucagon with an extension of twoamino acid in the C-terminal and glucagon(1-32) means human glucagonwith an extension of three amino acid in the C-terminal.

In other words, the peptide of the glucagon derivative is a glucagonpeptide which has a number of modifications of amino acid residues whencompared to human glucagon (SEQ ID NO: 1). These modifications mayrepresent, independently, one or more amino acid substitutions,additions, and/or deletions. For example, “[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28]-Glucagon amide” designates glucagon (SEQ ID NO: 1), whereinthe amino acid in position 2 has been substituted with Aib, the aminoacid in position 10 has been substituted with Leu, the amino acid inposition 16 has been substituted with Lys, the amino acid in position 20has been substituted with Arg, the amino acid in position 27 has beensubstituted with Leu, the amino acid in position 28 has been substitutedwith Ser, and the C-terminal carboxylic acid has been replaced with aC-terminal amide.

Analogues “comprising” certain specified changes may comprise furtherchanges, when compared to SEQ ID NO: 1. In a particular embodiment, theanalogue “has” the specified changes.

In one embodiment, the terms “peptide” and “analogue” (including e.g.“glucagon peptide”, “peptide analogue” and “glucagon analogue”) are usedinterchangeably herein and refer to the amino acid sequence of theglucagon derivative of the invention.

The expressions “position” or “corresponding position” may be used tocharacterise the site of change in an amino acid sequence by referenceto glucagon (SEQ ID NO: 1). The position, as well as the number ofchanges, are easily deduced, e.g. by simple handwriting and eyeballing.

The term “glucagon analogue” as used herein referring to the glucagonsequence wherein one or more amino acid residues of the native peptidehave been substituted by other amino acid residues and/or wherein one ormore amino acid residues have been deleted from the glucagon sequenceand/or wherein one or more amino acid residues have been added to thepeptide. Such addition or deletion of amino acid residues can take placeat the N-terminal of the peptide and/or at the C-terminal of thepeptide. A simple system is used to describe analogues. Formulae ofpeptide analogues and derivatives thereof are drawn using standardsingle letter or three letter abbreviations for amino acids usedaccording to IUPAC-IUB nomenclature. The term “analogue” as used hereinreferring to a polypeptide means a modified peptide wherein one or moreamino acid residues of the peptide have been substituted by other aminoacid residues and/or wherein one or more amino acid residues have beendeleted from the peptide and/or wherein one or more amino acid residueshave been added to the peptide. Such addition or deletion of amino acidresidues can take place at the N-terminal of the peptide and/or at theC-terminal of the peptide.

The term “polypeptide” and “peptide” as used herein means a compoundcomposed of at least five constituent amino acids connected by peptidebonds. The constituent amino acids may be from the group of the aminoacids encoded by the genetic code and they may be natural amino acidswhich are not encoded by the genetic code, as well as synthetic aminoacids. Natural amino acids which are not encoded by the genetic code aree.g. hydroxyproline, γ-carboxyglutamate, ornithine, phosphoserine,D-alanine and D-glutamine. Synthetic amino acids comprise amino acidsmanufactured by chemical synthesis, i.e. D-isomers of the amino acidsencoded by the genetic code such as D-alanine and D-leucine, Aib(α-aminoisobutyric acid), Acb (1-Aminocyclobutanecarboxylic acid), Acpr(1-Aminocyclopropanecarboxylic acid), Abu (α-aminobutyric acid), Tle(tert-butylglycine), β-alanine, 3-aminomethyl benzoic acid, anthranilicacid.

As is apparent from the above examples, amino acid residues may beidentified by their full name, their one-letter code, and/or theirthree-letter code. These three ways are fully equivalent. In oneembodiment peptide analogues and derivatives thereof are drawn usingstandard one-letter or three-letter codes according to IUPAC-IUBnomenclature. In the present context, common rules for peptidenomenclature based on the three or one letter amino acid code apply.Briefly, the central portion of the amino acid structure is representedby the three letter code (e.g. Ala, Lys) or one letter code (e.g. A, K)and L-configuration is assumed, unless D-configuration is specificallyindicated by “D-” followed by the three letter code (e.g. D-Ala, D-Lys).A substituent at the amino group replaces one hydrogen atom and its nameis placed before the three letter code, whereas a C-terminal substituentreplaces the carboxylic hydroxy group and its name appears after thethree letter code. For example, “acetyl-Gly-Gly-NH₂” representsCH₃—C(═O)—NH—CH₂—C(═O)—NH—CH₂—C(═O)—NH₂. Unless indicated otherwise,amino acids are connected to their neighbouring groups by amide bondsformed at the N-2 (α-nitrogen) atom and the C-1 (C═O) carbon atom.

The amino acid abbreviations used in the present context have thefollowing meanings:

Amino acid Description Acb 1-Aminocyclobutancarboxylic acid Acpr1-Aminocyclopropanecarboxylic acid Ado

Aib 2-Aminoisobutyric acid Ala Alanine Asn Asparagine Asp Aspartic acidArg Arginine Cit Citrulline Cys Cysteine Gln Glutamine Glu Glutamic acidγ-Glu

Gly Glycine His Histidine Hyp 4-hydroxyproline Ile Isoleucine LeuLeucine Lys Lysine Met Methionine Met(O)

Orn Ornithine Phe Phenylalanine Pro Proline Ser Serine Thr Threonine TyrTyrosine p(Tyr)

Trp Tryptophan Val Valine

Amino acid abbreviations beginning with D- followed by a three lettercode, such as D-Ser, D-His and so on, refer to the D-enantiomer of thecorresponding amino acid, for example D-serine, D-histidine and so on.

The term “glucagon amide”, means glucagon wherein the C-terminalcarboxylic acid has been replaced with a C-terminal amide.

The glucagon derivative may comprise an amino acid sequence of Formula Ihaving a total of up to 15 amino acid differences (also referred toherein as modifications) as compared to glucagon (SEQ ID NO: 1), forexample one or more additions, one or more deletions and/or one or moresubstitutions. In one embodiment the amino acid sequence of Formula Icomprises 3-15 amino acid residue modifications, such as substitutionsor additions, as compared to glucagon (SEQ ID NO: 1). In one embodimentthe amino acid sequence of Formula I comprises 4-15 or 5-15 amino acidresidue modifications, such as substitutions or additions, as comparedto glucagon (SEQ ID NO: 1). In one embodiment the amino acid sequence ofFormula I comprises up to 14, such as up to 13 or up to 12, amino acidresidue modifications, such as substitutions or additions, as comparedto glucagon (SEQ ID NO: 1). In one embodiment the amino acid sequence ofFormula I comprises up to 11, such as up to 10 or up to 9, amino acidresidue modifications, such as substitutions or additions, as comparedto glucagon (SEQ ID NO: 1). In one embodiment the amino acid sequence ofFormula I comprises up to 8, such as up to 7 or up to 6, amino acidresidue modifications, such as substitutions or additions, as comparedto glucagon (SEQ ID NO: 1).

In one embodiment the glucagon derivative is of Formula I as describedherein, wherein

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, or Ala;

X₂₀ represents Gln, Arg, Glu, or Lys;

X₂₁ represents Asp, Glu or Lys;

X₂₄ represents Gln, Ala, Arg, or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, or Lys;

X₂₉ represents Thr, Gly, or Lys; and

X₃₀ represents Lys, or X₃₀ is absent.

In one embodiment the glucagon derivative is of Formula I as describedherein, wherein

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, or Ala;

X₂₀ represents Gln, Arg, Glu, or Lys;

X₂₁ represents Asp, Glu or Lys;

X₂₄ represents Gln, Ala, Arg, or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, or Lys;

X₂₉ represents Thr, Gly, or Lys; and

X₃₀ represents Lys, or X₃₀ is absent.

In one embodiment the glucagon derivative is of Formula I as describedherein, wherein X₂ represents Aib, Acb or Acpr. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₂represents Aib. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₂ represents Acb. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₂represents Acpr. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₃ represents Gln or His. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₃ represents Gln. In one embodiment the glucagon derivative isof Formula I as described herein, wherein X₃ represents His. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₁₀ represents Leu, Ile or Val. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₁₀ representsLeu. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₁₀ is Ile. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₁₀ representsVal. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₁₂ represents Lys or Arg. In one embodimentthe glucagon derivative is of Formula I as described herein, wherein X₁₂represents Lys. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₁₂ represents Arg. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₁₅represents Asp or Glu. In one embodiment the glucagon derivative is ofFormula I as described herein, wherein X₁₅ represents Asp. In oneembodiment the glucagon derivative is as described herein, wherein X₁₅represents Glu. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₁₆ represents Ser, Ala, Leu, Thr, Glu,Aib, Ile, Val or Lys. In one embodiment the glucagon derivative is ofFormula I as described herein, wherein X₁₆ represents Ser, Ala, Leu,Thr, Glu, Aib, or Lys. In one embodiment the glucagon derivative is ofFormula I as described herein, wherein X₁₆ represents Ser, Ala, Leu,Thr, Glu or Lys. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₁₆ represents Ala, Leu, Thr, Glu or Lys.In one embodiment the glucagon derivative is of Formula I as describedherein, wherein X₁₆ represents Ser. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₁₆ representsAla. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₁₆ represents Leu. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₁₆represents Thr. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₁₆ represents Glu. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₁₆represents Lys. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₁₇ represents Arg or Lys. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₁₇ represents Arg. In one embodiment the glucagon derivative isof Formula I as described herein, wherein X₁₇ represents Lys. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₁₈ represents Arg, or Ala. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₁₈ representsArg. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₁₈ represents Ala. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₂₀represents Gln, Arg, Glu, Aib or Lys. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₂₀ representsGln, Arg, Glu, or Lys. In one embodiment the glucagon derivative is ofFormula I as described herein, wherein X₂₀ represents Gln. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₀ represents Arg. In one embodiment the glucagon derivative isof Formula I as described herein, wherein X₂₀ represents Glu. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₀ represents Lys. In one embodiment the glucagon derivative isof Formula I as described herein, wherein X₂₁ represents Asp, Glu orLys. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₂₁ represents Glu or Lys. In one embodimentthe glucagon derivative is of Formula I as described herein, wherein X₂₁represents Asp. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₂₁ represents Glu. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₂₁represents Lys. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₂₄ represents Gln, Ala, Arg, Glu, Aib orLys. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₂₄ represents Gln, Ala, Arg, or Lys. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₄ represents Gln. In one embodiment the glucagon derivative isof Formula I as described herein, wherein X₂₄ represents Ala. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₄ represents Arg. In one embodiment the glucagon derivative isof Formula I as described herein, wherein X₂₄ represents Lys. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₇ represents Met, Leu or Val. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₂₇ representsLeu or Val. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₂₇ represents Met. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₂₇represents Leu. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₂₇ represents Val. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₂₈represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys. In one embodimentthe glucagon derivative is of Formula I as described herein, wherein X₂₈represents Asn, Ser, or Lys. In one embodiment the glucagon derivativeis of Formula I as described herein, wherein X₂₈ represents Asn. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₈ represents Ser. In one embodiment the glucagon derivative isof Formula I as described herein, wherein X₂₈ represents Lys. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₉ represents Thr, Gly, Ser, Gln, Ala, Glu or Lys. In oneembodiment the glucagon derivative is of Formula I as described herein,wherein X₂₉ represents Thr, Gly, or Lys. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₂₉ representsGly or Lys. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₂₉ represents Thr. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₂₉represents Gly. In one embodiment the glucagon derivative is of FormulaI as described herein, wherein X₂₉ represents Lys. In one embodiment theglucagon derivative is of Formula I as described herein, wherein X₃₀represents Lys, or wherein X₃₀ is absent. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₃₀ representsLys. In one embodiment the glucagon derivative is of Formula I asdescribed herein, wherein X₃₀ is absent. In one embodiment the glucagonderivative is of Formula I as described herein, wherein X₂ representsAib; X₂₀ represents Arg; and X₂₁ represents Glu.

In one embodiment the glucagon derivative is of Formula I as describedherein, comprising an amino acid sequence with any one of the followingamino acid substitutions:

[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28];

[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28];

[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28];

[Aib2,Leu10,Arg20,Leu27,Lys28];

[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Arg20,Leu27,Ser28];

[Aib2,Leu10,Lys16,Lys17,Glu21,Leu27];

[Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29];

[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Lys16,Arg20,Glu21,Leu27];

[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29];

[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28];

[Aib2,Leu10,Lys16,Lys17,Ala8,Arg20,Glu21,Ala24,Leu27];

[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27];

[Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27];

[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29];

[Aib2,Leu10,Lys16,Glu21,Leu27];

[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28];

[Acb2,His3,Leu10,Glu15,Leu27,Lys28];

[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28];

[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28];

[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28];

[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28];

[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Leu16,Leu27,Lys28];

[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28];

[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28];

[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Glu15,Leu27,Lys28];

[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28];

[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29];

[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]; and

[Aib2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions:

[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28];[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28]; and[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with any one of the following amino acid substitutions:[Aib2,Leu10,Arg20,Leu27,Lys28];[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29]; and[Aib2,Leu10,Arg20,Leu27,Ser28]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with any one of the following amino acid substitutions:[Aib2,Leu10,Lys16,Lys17,Glu21,Leu27];[Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29]; and[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Aib2,Leu10,Lys16,Arg20,Glu21,Leu27];[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29]; and[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with any one of the following amino acid substitutions: [Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29];[Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29]; and[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Acb2,Leu10,Glu15,Glu16,Arg20,Leu27, Lys28];[Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27]; and[Aib2,Leu10,Lys16, Ala18,Arg20,Glu21,Ala24,Leu27]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27];[Aib2,Leu10,Glu15, Arg20,Glu21,Leu27,Lys28]; and[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Aib2,Leu10, Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21, Leu27,Lys28]; and[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Aib2,His3, Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];[Aib2,Leu10,Lys16,Glu21,Val27,Lys28, Gly29]; and[Aib2,Leu10,Lys16,Glu21,Leu27]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with any one of the following amino acid substitutions:[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28]; and[Aib2,His3,Leu10,Glu15, Lys16,Arg20,Glu21,Ala24,Leu27,Ser28]. In oneembodiment the glucagon derivative is of Formula I as described herein,comprising an amino acid sequence with any one of the following aminoacid substitutions: [Acb2,His3,Leu10,Glu15,Leu27,Lys28]; [Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; and[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28].

In one embodiment the glucagon derivative is of Formula I as describedherein, comprising an amino acid sequence with any one of the followingamino acid substitutions: [Aib2, Leu10,Leu16,Arg20,Glu21,Leu27,Lys29];[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29]; and[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Acb2,Leu10,Glu15,Arg20,Glu21,Leu27, Lys28];[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]; and [Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28]. In one embodiment the glucagon derivative is of Formula Ias described herein, comprising an amino acid sequence with any one ofthe following amino acid substitutions:[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]; [Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28]; and[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28];[Aib2,Leu10,Leu16,Leu27,Lys28]; and[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with any one of the following amino acid substitutions:[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28];[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28]; and[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with any one of the following amino acid substitutions:[Acb2,Leu10, Arg12,Glu15,Arg20,Glu21,Leu27,Lys28];[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28]; and[Acb2,Leu10,Glu15,Leu27,Lys28]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with any one of the following amino acid substitutions:[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28];[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29];[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]; and[Aib2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Aib2,Leu10,Ala16, Arg20,Leu27,Lys28];[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; and[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21, Leu27,Lys28]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with any one of the following amino acidsubstitutions: [Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];[Acb2,Leu10,Glu15, Arg20,Glu21,Leu27,Lys28]; and[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]. In one embodiment the glucagonderivative is of Formula I as described herein, comprising an amino acidsequence with the following amino acid substitutions:[Aib2,Leu10,Glu15,Lys17, Arg20,Ser21,Leu27,Lys28]. In one embodiment theglucagon derivative is of Formula I as described herein, comprising anamino acid sequence with the following amino acid substitutions:[Aib2,Val10,Ala16,Leu27,Lys28].

In one embodiment the glucagon derivative is of Formula I as describedherein, the amino acid sequence comprises a lysine residue at one ormore of positions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30. In oneembodiment the glucagon derivative is of Formula I as described herein,the amino acid sequence comprises a lysine residue at one, two or threeof positions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30. In oneembodiment the glucagon derivative is of Formula I as described herein,the amino acid sequence comprises a lysine residue at one or two ofpositions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30. In oneembodiment the glucagon derivative is of Formula I as described herein,the amino acid sequence comprises a lysine residue at two of positions12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30; in particular inpositions 12 and 28. In one embodiment the glucagon derivative is ofFormula I as described herein, the amino acid sequence comprises alysine residue at position 12, 16, 17, 18, 20, 21, 24, 28, 29, or 30.

In one embodiment the amino acid sequence of Formula I consists ofFormula I. In one embodiment X₂ is Aib, Acb, or Acpr. In one embodimentX₃ represents His. In one embodiment X₁₀ represents Leu or Val. In oneembodiment X₁₂ represents Arg. In one embodiment X₁₅ represents Glu. Inone embodiment X₁₆ represents Ala, Glu, Leu, Lys, Thr or Aib, such asAla, Glu or Leu, or such as Lys, Thr or Aib. In one embodiment X₁₇represents Lys. In one embodiment X₁₈ represents Ala. In one embodimentX₂₀ represents Arg, Lys or Glu. In one embodiment X₂₁ represents Lys,Glu or Ser. In one embodiment X₂₄ represents Ala, Arg or Lys. In oneembodiment X₂₇ represents Leu or Val. In one embodiment X₂₈ representsLys or Ser. In one embodiment X₂₉ represents Lys or Gly. In oneembodiment X₃₀ represents Lys or X₃₀ is absent.

Glucagon Derivative

The invention relates to glucagon derivatives. The term “glucagonderivative” as used herein means chemically modified glucagon or ananalogue thereof, wherein at least one substituent is not present in theunmodified peptide or an analogue thereof, i.e. where glucagon has beencovalently modified. Typical modifications are amides, carbohydrates,alkyl groups, acyl groups, esters and the like. The term “derivative” asused herein in relation to a peptide means a chemically modified peptideor an analogue thereof, wherein at least one substituent is not presentin the unmodified peptide or an analogue thereof, i.e. a peptide whichhas been covalently modified. Typical modifications are amides,carbohydrates, alkyl groups, acyl groups, esters and the like. In oneembodiment the term “glucagon derivative” as used herein means glucagonderivative, glucagon compound, compound according to the invention,compound of the invention, compound of Formula I, a glucagon analogue, aglucagon derivative or a derivative of a glucagon analogue humanglucagon, human glucagon(1-29), glucagon(1-30), glucagon(1-31),glucagon(1-32) as well as analogues, and fusion peptides thereof, whichmaintain glucagon activity.

In one embodiment the glucagon derivative comprises a substituentcovalently attached to the glucagon analogue via the side chain of alysine. The term “substituent” as used herein, means a chemical moietyor group replacing a hydrogen.

The term “distal” as used herein, means most remote (terminal) from thepoint of attachment.

In one embodiment the term “negatively charged moiety” as used herein,means a negatively chargeable chemical moiety, such as, but not limitedto, a carboxylic acid (e.g.

Glu, gamma-Glu, Asp or beta-Asp), sulphonic acid or a tetrazole moiety.In one embodiment the term “negatively charged moiety” as used herein,means a negatively chargeable chemical moiety such as, but not limitedto a carboxylic acid, sulphonic acid or a tetrazole moiety. In oneembodiment the substituent has three to ten negatively charged moieties.In one embodiment the substituent has 3, 4, 5, 6, 7, 8, 9 or 10negatively charged moieties. In one embodiment the substituent isnegatively charged at physiological pH. In one embodiment the number of“negatively charged moieties” is determined at physiological pH (pH7.4). In one embodiment the “negatively charged moiety” is a carboxylicacid group.

The term “lipophilic moiety” as used herein, means an aliphatic orcyclic hydrocarbon moiety with more than 6 and less than 30 carbonatoms, wherein said hydrocarbon moiety may contain additionalsubstituents.

The term “albumin binding residue” as used herein means a residue whichbinds non-covalently to human serum albumin. The albumin binding residueattached to the therapeutic polypeptide typically has an affinity below10 μM to human serum albumin and preferably below 1 μM. A range ofalbumin binding residues are known among linear and branchedlipohophillic moieties containing 4-40 carbon atoms.

The term “protracted effect” of the compounds of the invention meansthat the period of time in which they exert a biological activity isprolonged.

In the present context, the term “agonist” is intended to indicate asubstance (ligand) that activates the receptor type in question.

In one embodiment the symbol “*” when used herein in a drawing of achemical structure represents the point of attachment to theneighbouring position in the derivative.

In one embodiment the glucagon derivative of Formula I as describedherein, wherein a substituent comprising a lipophilic moiety and threeor more negatively charged moieties, wherein one of said negativelycharged moieties is distal of a lipophilic moiety, and wherein saidsubstituent is attached at the epsilon position of a Lys in one of thefollowing amino acid positions of said glucagon derivative: 16, 17, 18,20, 21, 24, 28, 29, and/or 30. In one embodiment the substituentcomprising a lipophilic moiety and at least three negatively chargedmoieties, is attached at the epsilon position of a lysine residue inposition 16, 21, 24, 28, 29, or 30. In one embodiment the substituentcomprising a lipophilic moiety and at least three negatively chargedmoieties, is attached at the epsilon position of a lysine residue inposition 16, 21, 24, 28, 29, or 30. In one embodiment the substituentcomprising a lipophilic moiety and at least three negatively chargedmoieties, is attached at the epsilon position of a lysine residue inposition 16, 24, 28, 29 or 30. In one embodiment the substituentcomprising a lipophilic moiety and at least three negatively chargedmoieties, is attached at the epsilon position of a lysine residue inposition 24, 28, 29 or 30. In one embodiment the substituent comprisinga lipophilic moiety and at least three negatively charged moieties, isattached at the epsilon position of a lysine residue in position 28, 29and 30. In one embodiment the substituent comprising a lipophilic moietyand at least three negatively charged moieties, is attached at theepsilon position of a lysine residue in position 28.

In one embodiment the substituent comprising a lipophilic moiety andthree or more negatively charged moieties is a substituent of FormulaII: Z¹-Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-(II), wherein,

Z¹-represents a structure of Formula IIa;

wherein n is 6-20; and the symbol * represents the attachment point tothe nitrogen of the neighbouring linking group; and

Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-represents a linking group, wherein each ofZ₂ to Z₁₀ individually are represented by any one of the following aminoacid residues: Glu, γGlu, Gly, Ser, Ala, Thr or Ado; or one or more ofresidues Z² to Z¹⁰ are absent; provided, however, that at least two ofresidues Z² to Z¹⁰ are present; and

wherein Z¹-Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-together contains at least threenegative charges; and wherein said substituent is attached at theepsilon position of a Lys residue according to Formula I.

In one embodiment the glucagon derivative is of Formula I as describedherein, wherein Z¹ of Formula II represents a structure according toFormula IIa:

wherein n represents an integer in the range of from 6 to 20; thesymbol * represents the attachment point to the nitrogen of theneighbouring group; and wherein Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, Z₁₀individually are represented by the following amino acids: Glu, γGlu,Gly, Ser, Ala, Thr and Ado; or one or more of Z₂, Z₃, Z₄, Z₅, Z₆, Z₇,Z₈, Z₉, Z₁₀ may be absent; provided, however, that at least two ofresidues Z₂ to Z₁₀ are present; wherein Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀together contains at least three negative charges; and wherein saidsubstituent is attached at the epsilon position of a Lys residueaccording to Formula I, herein.

In one embodiment n in Z¹ of Formula IIa is (i.e. represents) 14, 16 or18. In one embodiment n in Z¹ of Formula IIa is 14. In one embodiment nin Z¹ of Formula IIa is 16. In one embodiment n in Z¹ of Formula IIa is18.

In one embodiment Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-represents a linkinggroup, wherein each of Z₂ to Z₁₀ individually are represented by any oneof the following amino acid residues: Glu, γGlu, Gly, Ser, or Ado (suchas Glu, γGlu, or Gly, or such as Ser, or Ado); or one or more ofresidues Z² to Z¹⁰ are absent; provided, however, that at least two ofresidues Z² to Z¹⁰ are present.

In one embodiment the substituent represents a structure according toany one of the following nine formulas (Chem.A-Chem.I), wherein *indicates the point of attachment to the nitrogen atom of the epsilonposition of a Lys residue of Formula I:

In one embodiment the substituent represents a structure according tothe formula Chem.J, wherein * indicates the point of attachment to thenitrogen atom of the epsilon position of a Lys residue of Formula I:

In one embodiment the substituent represents a structure according toany one of formulas Chem.A-Chem.I, as described herein, wherein *indicate the point of attachment to the nitrogen atom of the epsilonposition of a Lys residue according to Formula I. In one embodiment thesubstituent represents a structure according to the formula Chem.J, asdescribed herein, wherein * indicate the point of attachment to thenitrogen atom of the epsilon position of a Lys residue according toFormula I. In one embodiment the substituent represents a structureaccording to the formula Chem.B, Chem.C, or Chem.H, described herein,wherein * indicate the point of attachment to the nitrogen atom of theepsilon position of a Lys residue according to Formula I.

In one embodiment Z₁ represents a structure according to the FormulaIIa;

wherein n in Formula IIa is 6-20, and the symbol * represents theattachment point to the nitrogen of the neighbouring group and whereinZ₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, Z₁₀ individually are represented by thefollowing amino acids: Glu, γGlu, Gly, Ser, Ala, Thr, Ado or is absent.In one embodiment Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, Z₁₀ individually arerepresented by the following amino acids: Glu, γGlu, Gly, Ser, Ado or isabsent.

In one embodiment one of said negatively charged moieties is distal ofsaid lipophilic moiety. In one embodiment the substituent bindsnon-covalently to albumin.

In one embodiment the glucagon derivative isN^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε21)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε24)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(α)-([Aib2,Leu10,Arg20,Leu27,Ser28]-Glucagonyl)-N{Epsilon}[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]Lysamide

In one embodiment the glucagon derivative isN^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Glu21,Leu27]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Leu27]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys6,Glu20,Leu27,Ser28,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]-acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Leu27]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε16)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,His3,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxy-propanoyl]amino]acetyl]-[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagon amide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl][Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN²-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Acb2,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu2,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Ser21,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Ala16,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Leu16,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

In one embodiment the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Leu16,Leu27,Lys28]-Glucagonamide

Pharmaceutically Acceptable Salt, Amide, or Ester

The derivatives, analogues and intermediate products of the inventionmay be in the form of a pharmaceutically acceptable salt, amide, orester.

Salts are e.g. formed by a chemical reaction between a base and an acid,e.g.:

2NH₃+H₂SO₄→(NH₄)₂SO₄.

The salt may be a basic salt, an acid salt, or it may be neither nor(i.e. a neutral salt). Basic salts produce hydroxide ions and acid saltshydronium ions in water.

The salts of the derivatives or analogues of the invention may be formedwith added cations or anions between anionic or cationic groups,respectively. These groups may be situated in the peptide moiety, and/orin the side chain of the analogues of the invention.

Non-limiting examples of anionic groups of the derivatives or analoguesof the invention include free carboxylic groups in the side chain, ifany, as well as in the peptide moiety. The peptide moiety often includesfree carboxylic groups at internal acid amino acid residues such as Aspand Glu.

Non-limiting examples of cationic groups in the peptide moiety includethe free amino group at the N-terminus, if present, as well as any freeamino group of internal basic amino acid residues such as His, Arg, andLys.

The ester of the derivatives or analogues of the invention may e.g. beformed by the reaction of a free carboxylic acid group with an alcoholor a phenol, which leads to replacement of at least one hydroxyl groupby an alkoxy or aryloxy group

The ester formation may involve the free carboxylic group at theC-terminus of the peptide, and/or any free carboxylic group in the sidechain.

The amide of the derivatives or analogues of the invention may e.g. beformed by the reaction of a free carboxylic acid group with an amine ora substituted amine, or by reaction of a free or substituted amino groupwith a carboxylic acid.

The amide formation may involve the free carboxylic group at theC-terminus of the peptide, any free carboxylic group in the side chain,the free amino group at the N-terminus of the peptide, and/or any freeor substituted amino group of the peptide in the peptide and/or the sidechain.

In one embodiment the derivative or peptide is in the form of apharmaceutically acceptable salt. In another embodiment, the derivativeor peptide is in the form of a pharmaceutically acceptable amide,preferably with an amide group at the C-terminus of the peptide. In astill further embodiment, the derivative or peptide is in the form apharmaceutically acceptable ester.

The term “pharmaceutical composition” as used herein means a productcomprising an active compound, e.g. the glucagon derivative of theinvention, or a salt thereof together with pharmaceutical excipientssuch as buffer, preservative, and optionally a tonicity modifier and/ora stabilizer. Thus a pharmaceutical composition is also known in the artas a pharmaceutical formulation.

In the present context, the term “pharmaceutically acceptable salt” isintended to indicate a salt which is not harmful to the patient. Suchsalts include pharmaceutically acceptable acid addition salts,pharmaceutically acceptable metal salts, ammonium and alkylated ammoniumsalts. Acid addition salts include salts of inorganic acids as well asorganic acids.

The term “excipient” as used herein means the chemical compounds whichare normally added to pharmaceutical compositions, e.g. buffers,tonicity agents, preservatives and the like.

As use herein, the term “therapeutically effective amount” of acompound, e.g. the glucagon derivative of the invention, refers to anamount sufficient to cure, alleviate or partially arrest the clinicalmanifestations of a given disease and/or its complications. An amountadequate to accomplish this is defined as a “therapeutically effectiveamount”. Effective amounts for each purpose will depend on the severityof the disease or injury, as well as on the weight and general state ofthe subject. It will be understood that determination of an appropriatedosage may be achieved using routine experimentation, by constructing amatrix of values and testing different points in the matrix, all ofwhich is within the level of ordinary skill of a trained physician orveterinarian.

The terms “treatment”, “treating” and other variants thereof as usedherein refer to the management and care of a patient for the purpose ofcombating a condition, such as a disease or a disorder. The terms areintended to include the full spectrum of treatments for a givencondition from which the patient is suffering, such as administration ofthe active compound(s) in question to alleviate symptoms orcomplications thereof, to delay the progression of the disease, disorderor condition, to cure or eliminate the disease, disorder or condition,and/or to prevent the condition, in that prevention is to be understoodas the management and care of a patient for the purpose of combating thedisease, condition, or disorder, and includes the administration of theactive compound(s) in question to prevent the onset of symptoms orcomplications. The patient to be treated is preferably a mammal, inparticular a human being, but treatment of other animals, such as dogs,cats, cows, horses, sheep, goats or pigs, is within the scope of theinvention.

The term “diabetes” or “diabetes mellitus” includes type 1 diabetes,type 2 diabetes, gestational diabetes (during pregnancy) and otherstates that cause hyperglycaemia. The term is used for a metabolicdisorder in which the pancreas produces insufficient amounts of insulin,or in which the cells of the body fail to respond appropriately toinsulin thus preventing cells from absorbing glucose. As a result,glucose builds up in the blood.

Type 1 diabetes, also called insulin-dependent diabetes mellitus (IDDM)and juvenile-onset diabetes, is caused by beta-cell destruction, usuallyleading to absolute insulin deficiency.

Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus(NIDDM) and adult-onset diabetes, is associated with predominant insulinresistance and thus relative insulin deficiency and/or a predominantlyinsulin secretory defect with insulin resistance.

The term “euglycemia” as used herein means normal concentration ofglucose in the blood. This is also referred to as normoglycemia.

The term “obesity” implies an excess of adipose tissue. When energyintake exceeds energy expenditure, the excess calories are stored inadipose tissue, and if this net positive balance is prolonged, obesityresults, i.e. there are two components to weight balance, and anabnormality on either side (intake or expenditure) can lead to obesity.In this context, obesity is best viewed as any degree of excess adiposetissue that imparts a health risk. The distinction between normal andobese individuals can only be approximated, but the health risk impartedby obesity is probably a continuum with increasing adipose tissue.However, in the context of the invention, individuals with a body massindex (BMI=body weight in kilograms divided by the square of the heightin meters) above 25 are to be regarded as obese.

In the case of administration of a glucagon derivative of the invention,optionally in combination with one or more additional therapeuticallyactive compounds or substances as disclosed above, for a purpose relatedto treatment or prevention of obesity or overweight, i.e. related toreduction or prevention of excess adiposity, it may be of relevance toemploy such administration in combination with surgical intervention forthe purpose of achieving weight loss or preventing weight gain, e.g. incombination with bariatric surgical intervention.

Examples of frequently used bariatric surgical techniques include, butare not limited to, the following: vertical banded gastroplasty (alsoknown as “stomach stapling”), wherein a part of the stomach is stapledto create a smaller pre-stomach pouch which serves as a new stomach;gastric banding, e.g. using an adjustable gastric band system (such asthe Swedish Adjustable Gastric Band (SAGB), the LAP-BAND™ or theMIDband™), wherein a small pre-stomach pouch which is to serve as a newstomach is created using an elastomeric (e.g. silicone) band which canbe adjusted in size by the patient; and gastric bypass surgery, e.g.“Roux-en-Y” bypass wherein a small stomach pouch is created using astapler device and is connected to the distal small intestine, the upperpart of the small intestine being reattached in a Y-shapedconfiguration.

The administration of a glucagon derivative of the invention (optionallyin combination with one or more additional therapeutically activecompounds or substances as disclosed above) may take place for a periodprior to carrying out the bariatric surgical intervention in questionand/or for a period of time subsequent thereto. In many cases it may bepreferable to begin administration of a compound of the invention afterbariatric surgical intervention has taken place.

The compounds of the invention and anti-obesity or anti-diabetic agents,as defined in the present specification, may be administeredsimultaneously or sequentially. The factors may be supplied insingle-dosage form wherein the single-dosage form contains bothcompounds, or in the form of a kit-of-parts comprising a preparation ofa compound of the invention as a first unit dosage form and apreparation of an anti-obesity or anti-diabetic agent as a second unitdosage form. Whenever a first or second or third, etc., unit dose ismentioned throughout this specification this does not indicate thepreferred order of administration, but is merely done for conveniencepurposes.

By “simultaneous” dosing of a preparation of a compound of the inventionand a preparation of anti-obesity or anti-diabetic agents is meantadministration of the compounds in single-dosage form, or administrationof a first agent followed by administration of a second agent with atime separation of no more than 15 minutes, preferably 10, morepreferred 5, more preferred 2 minutes. Either factor may be administeredfirst.

By “sequential” dosing is meant administration of a first agent followedby administration of a second agent with a time separation of more than15 minutes. Either of the two unit dosage form may be administeredfirst. Preferably, both products are injected through the sameintravenous access.

As already indicated, in all of the therapeutic methods or indicationsdisclosed above, a compound of the invention may be administered alone.However, it may also be administered in combination with one or moreadditional therapeutically active agents, substances or compounds,either sequentially or concomitantly.

A typical dosage of a compound of the invention when employed in amethod according to the invention is in the range of from about 0.0001to about 100 mg/kg body weight per day, preferably from about 0.001 toabout 10 mg/kg body weight, more preferably from about 0.001 to about 5mg/kg body weight per day, e.g. from about 0.001 to about 10 mg/kg bodyweight per day or from about 0.001 to about 5 mg/kg body weight per dayadministered in one or more doses, such as from 1 to 3 doses. The exactdosage will depend upon the frequency and mode of administration, thesex, age, weight and general condition of the treated subject, thenature and severity of the condition treated, any concomitant diseasesto be treated and other factors evident to those skilled in the art.

Compounds of the invention comprise compounds that are believed to bewell-suited to administration with longer intervals than, for example,once daily, thus, appropriately formulated compounds of the inventionmay be suitable for e.g. twice-weekly or once-weekly administration by asuitable route of administration, such as one of the routes disclosedherein.

As described above, compounds of the invention may be administered orapplied in combination with one or more additional therapeuticallyactive compounds or substances, and suitable additional compounds orsubstances may be selected, for example, from antidiabetic agents,antihyperlipidemic agents, antiobesity agents, antihypertensive agentsand agents for the treatment of complications resulting from, orassociated with, diabetes.

Preparation of Derivatives of Glucagon Peptides

In one embodiment the invention relates to a process for making aglucagon derivative according the invention. The derivative of theinvention may be prepared by the method described below.

SPPS General Methods

The Fmoc-protected amino acid derivatives to be used may be the standardrecommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-lle-OH,Fmoc-Leu-OH, Fmoc-Lys(BOC)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(BOC)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem,Iris Biotech, or NovabioChem. Where nothing else is specified thenatural L-form of the amino acids are used. The N-terminal amino acid isBoc protected at the alpha amino group (e.g. Boc-His(Boc)-OH, orBoc-His(Trt)-OH for peptides with His at the N-terminus). SPPS may beperformed using Fmoc based chemistry on a Prelude Solid Phase PeptideSynthesizer from Protein Technologies (Tucson, Ariz. 85714 U.S.A.). Asuitable resin for the preparation of C-terminal peptide amides isH-Rink Amide-ChemMatrix resin (loading e.g. 0.52 nmol/g) or Rink AmideAM polystyrene resin (Novabiochem, loading e.g. 0.62 mmol/g) or thelike. Fmoc-deprotection is achieved with 20% piperidine in NMP. Peptidecouplings are performed by using either DIC/HOAt/collidine or DIC/OxymaPure/collidine without preactivation. Amino acid/HOAt or aminoacid/Oxyma Pure solutions (0.3 M/0.3 M in NMP at a molar excess of 3-10fold) are added to the resin followed by the same molar equivalent ofDIC (3 M in NMP) followed by collidine (3 M in NMP). For example, thefollowing amounts of 0.3 M amino acid/HOAt solution can be used percoupling for the following scale reactions: Scale/mL, 0.05 mmol/1.5 mL,0.10 mmol/3.0 mL, 0.25 mmol/7.5 mL. The Mtt group may be removed bywashing the resin with HFIP/DCM (75:25) (2×2 min), washing with DCM andsuspending the resin in HFIP/DCM (75:25)(2×20 min) and subsequentlywashing in sequence with Piperidine/NMP (20:80), DCM(1×), NMP(1×),DCM(1×), NMP(1×).

Attachment of the Substituent

The substituent can be introduced in a stepwise procedure by the Preludepeptide synthesizer as described above using suitably protected buildingblocks, such as the standard amino acids described above,Fmoc-8-amino-3,6-dioxaoctanoic acid and Fmoc-Glu-OtBu. Introduction ofthe fatty acid moiety can be achieved using a building block, such as,but not limited to, octadecanedioic acid mono-tert-butyl-ester. Aftereach coupling step, unreacted peptide intermediate can be capped usingacetic acid anhydride and collidine in excess (>10 eq.).

The introduction of a substituent on the epsilon-nitrogen of a lysine isachieved using a Lysine protected with Mtt (Fmoc-Lys(Mtt)-OH). The MTTgroup can be removed using standard procedure such as Alternatively, theepsilon-nitrogen of a lysine could be protected with an ivDde group(Fmoc-Lys(ivDde)-OH). The incorporation of gamma-Glu moieties in thesubstituent may be achieved by coupling with the amino acidFmoc-Glu-OtBu.

Introduction of each moiety in the substituent can be achieved usingprolonged coupling time (1×6 hours) followed by capping with aceticanhydride or alternatively acetic acid/DIC/HOAt/collidine.

Cleavage from the Resin

After synthesis the resin is washed with DCM, and the peptide is cleavedfrom the resin by a 2-3 hour treatment with TFA/TIS/water (95/2.5/2.5)followed by precipitation with diethylether. The precipitate is washedwith diethylether.

Purification and Quantification

The crude peptide is dissolved in a suitable mixture of water and MeCN,such as water/MeCN (4:1), and purified by reversed-phase preparativeHPLC (Waters Deltaprep 4000 or Gilson) on a column containing C18-silicagel. Elution is performed with an increasing gradient of MeCN in watercontaining 0.1% TFA. Relevant fractions are checked by analytical HPLCor UPLC. Fractions containing the pure target peptide are mixed andconcentrated under reduced pressure. The resulting solution is analyzed(HPLC, LCMS) and the product (i.e. the derivative) is quantified using achemiluminescent nitrogen specific HPLC detector (Antek 8060 HPLC-CLND)or by measuring UV-absorption at 280 nm. The product is dispensed intoglass vials. The vials are capped with Millipore glassfibre prefilters.Freeze-drying affords the peptide trifluoroacetate as a white solid.

FUNCTIONAL PROPERTIES

In a first functional embodiment, the peptides of the invention are ableto bind to both the GLP-1 receptor and glucagon receptor with goodaffinity. In a second functional embodiment, the peptides of theinvention preferably are GLP-1 and glucagon receptor agonists as isreflected by their potency on both receptors. Also, in a thirdfunctional embodiment, they have improved pharmacokinetic properties.Also, or alternatively, in a fourth functional embodiment, they havegood biophysical properties.

In one embodiment the invention provides novel GLP-1/glucagonRco-agonists with an improved receptor potency and/or affinity on theGLP-1 receptor or the glucagon receptor or on both receptors. In anotherembodiment, the invention provides novel GLP-1/glucagonR co-agonistswith improved stability. In another embodiment, the invention providesnovel GLP-1/glucagonR co-agonists with improved solubility. In anotherembodiment, the invention provides novel GLP-1/glucagonR co-agonistswith increased half-life.

The potency (i.e. receptor activation) and affinity (i.e. receptorbinding) on the glucagon and GLP-1 receptors may be determined accordingto the assays described in Examples 74-75 herein.

The solubility of the compounds of the invention at different pH valuesmay be measured as described in the Functional Properties sectionherein.

The physical stability of the compounds of the invention may be measuredby the Thioflavin (ThT) fibrillation assay described in Example 76herein.

The chemical stability of the glucagon derivatives or analogues of theinvention may be determined as described in Example 79 herein.

The peptides of the invention also have prolonged in vivo half-life. Thehalf-life of the glucagon derivatives or analogues of the invention maybe determined in a pharmacokinetic study in various species, includingmice (as described in Example 78 herein), rats and minipigs.

The effect of the glucagon derivatives of the invention on the reductionof body weight may be determined in DIO mice as described in Example 77herein.

In one embodiment the glucagon derivative of the invention is asselective for glucagon receptor as for GLP-1 receptor.

GLP-1/Glucagon Receptor Co-Agonists

In one embodiment the invention provides novel stable and protractedGLP-1/glucagon receptor co-agonists. A GLP-1/glucagon receptorco-agonist may be defined as a peptide that is able to activate both theGLP-1 and the glucagon receptors.

The derivatives of the invention show an EC₅₀ below 1 nM on the GLP-1receptor and an EC₅₀ below 10 nM on the glucagon receptor, or below 100pM on the GLP-1 receptor and below 100 pM on the glucagon receptor, orbelow 50 pM on the GLP-1 receptor and below 100 pM on the glucagonreceptor, or below 10 pM on the GLP-1 receptor and below 50 pM on theglucagon receptor. As mentioned, the receptor potencies may bedetermined using the assay described in Example 74 herein.

In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<100 pM and an EC₅₀<1 nM on the glucagon receptor.In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<50 pM and an EC₅₀<1 nM on the glucagon receptor.In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<1 nM on the glucagon receptor.In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<50 pM and an EC₅₀<100 pM on the glucagon receptor.In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<100 pM on the glucagon receptor.In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<50 pM on the glucagon receptor.In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<1 pM and an EC₅₀<50 pM on the glucagon receptor.In one embodiment the glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<10 pM on the glucagon receptor.In one embodiment the glucagon derivative is a GLP-1/glucagon co-agonistwith an EC₅₀ on the GLP-1 receptor<the EC₅₀ on the glucagon receptor. Inone embodiment the glucagon derivative is a GLP-1/glucagon co-agonistwith an EC₅₀ on the GLP-1 receptor (e.g. in pM) greater than the EC₅₀ onthe glucagon receptor (e.g. in pM).

Glucagon Receptor Agonist

A receptor agonist may be defined as a peptide that binds to a receptorand elicits a response typical of the natural ligand. The term “glucagonagonist” as used herein refers to any glucagon derivative which binds toand fully or partially activates the human glucagon receptor. In oneembodiment, the “glucagon agonist” is any glucagon derivative thatactivates the glucagon receptor, with a potency (EC₅₀) below 10 nM orbelow 1 nM or 100 pM or below 10 pM, as measured by the assay describedin Example 74 herein.

In one embodiment the glucagon derivative is an agonist of the glucagonreceptor. In one embodiment the glucagon derivative is an agonist of theglucagon receptor, with an EC₅₀<10 nM. In one embodiment the glucagonderivative is an agonist of the glucagon receptor, with an EC₅₀<1 nM. Inone embodiment the glucagon derivative is an agonist of the glucagonreceptor, with an EC₅₀<100 pM. In one embodiment the glucagon derivativeis an agonist of the glucagon receptor, with an EC₅₀<10 pM. The potency,i.e. EC₅₀, of the glucagon derivative on the glucagon receptor may bedetermined by the assay described in Example 74 herein.

GLP-1 Receptor Agonist

A receptor agonist may be defined as a peptide that binds to a receptorand elicits a response typical of the natural ligand. Thus, for example,a “GLP-1 receptor agonist” or “GLP-1 receptor agonist peptide” may bedefined as a compound which is capable of binding to the GLP-1 receptorand capable of activating it.

The co-agonist peptides of the invention have GLP-1 activity. This termrefers to the ability to bind to the GLP-1 receptor and initiate asignal transduction pathway resulting in insulinotropic action or otherphysiological effects as is known in the art. The derivatives of theinvention shows an EC₅₀ below 1 nM or below 100 pM or below 50 pM orbelow 10 pM using the assay described in Example 74 herein.

In one embodiment the glucagon derivative is an agonist of the GLP-1receptor. In one embodiment the glucagon derivative is an agonist of theGLP-1 receptor, with an EC₅₀<100 pM. In one embodiment the glucagonderivative is an agonist of the GLP-1 receptor, with an EC₅₀<50 pM. Inone embodiment the glucagon derivative is an agonist of the GLP-1receptor, with an EC₅₀<10 pM. The potency, i.e. EC₅₀, of the glucagonderivative on the GLP-1 receptor may be determined by the assaydescribed in Example 74 herein.

Biological Activity—In Vitro Affinity and Potency

In one embodiment affinity refers to in vitro binding affinity, i.e.performance in a GLP-1 receptor binding affinity assay and in a glucagonreceptor binding affinity assay, more in particular to the capability ofbinding the human GLP-1 receptor and to the human glucagon receptor. Thebinding affinity of the human GLP-1 receptor may be measured in abinding assay, e.g. in a stably transfected BHK cell line that expressesthe human GLP-1 receptor. Radioactively labelled GLP-1 binds to thereceptor and may be displaced competitively by a compound. Binding ofradioligand may be determined in the presence of scintillation proximityassay (SPA) beads which bind to cell membranes and when radioactivity isclose to the bead it produces light which is measured and is a measureof the in vitro binding affinity. One non-limiting example of such anassay is described in Example 75 herein. The binding affinity of thehuman glucagon receptor may be measured in a binding affinity assay,e.g. in a stably transfected BHK cell line that expresses the humanglucagon receptor. Radioactively-labelled glucagon binds to the receptorand may be displaced competitively by a compound. Binding of radioligandmay be determined in the presence of scintillation proximity assay (SPA)beads which bind to cell membranes and when radioactivity is close tothe bead it produces light which is measured and is a measure of the invitro binding affinity.

The term half maximal inhibitory concentration (IC₅₀) generally refersto the concentration of competing compound which displaces 50% of thespecific binding of the radioligand binding corresponding to halfwaybetween the baseline and maximum, by reference to the dose responsecurve. IC₅₀ is used as a measure of the binding affinity of a compoundand represents the concentration where 50% of its maximal binding isobserved.

The in vitro binding of the peptides of the invention may be determinedas described above, and the IC₅₀ of the peptide in question determined.The lower the IC₅₀ value, the better the binding affinity.

The affinity, i.e. IC₅₀, of the glucagon derivative on the GLP-1receptor may be determined by the assay described in Example 75 herein.In another embodiment, the peptide of the invention has an in vitrobinding affinity on the GLP-1 receptor determined using the method ofExample 75 herein corresponding to an IC₅₀ at or below 100 nM, morepreferably below 10 nM, even more preferably below 5 nM, or mostpreferably below 1 nM.

The affinity, i.e. IC₅₀, of the glucagon derivative on the glucagonreceptor may be determined by the assay described in Example 75 herein.In another embodiment, the peptide of the invention has an in vitrobinding affinity on the glucagon receptor determined using the method ofExample 75 herein corresponding to an IC₅₀ at or below 100 nM, or below50 nM or below 10 nM

In one embodiment potency refers to in vitro potency, i.e. performancein a functional GLP-1 receptor assay and glucagon receptor assay, morein particular to the capability of activating the human GLP-1 receptorand the human glucagon receptor. The response of the human GLP-1receptor may be measured in a reporter gene assay, e.g. in a stablytransfected BHK cell line that expresses the human GLP-1 receptor andcontains the DNA for the cAMP response element (CRE) coupled to apromoter and the gene for firefly luciferase (CRE luciferase). When cAMPis produced as a result of activation of the GLP-1 receptor this in turnresults in the luciferase being expressed. Luciferase expression may bedetermined by adding luciferin, which by the enzyme is converted tooxyluciferin and produces bioluminescence, which is measured and is ameasure of the in vitro potency. One non-limiting example of such anassay is described in Example 74 herein. The response of the humanglucagon receptor may be measured in a reporter gene assay, e.g. in astably transfected BHK cell line that expresses the human glucagonreceptor and contains the DNA for the cAMP response element (CRE)coupled to a promoter and the gene for firefly luciferase (CREluciferase). When cAMP is produced as a result of activation of theglucagon receptor this in turn results in the luciferase beingexpressed. Luciferase expression may be determined by adding luciferin,which by the enzyme is converted to oxyluciferin and producesbioluminescence, which is measured and is a measure of the in vitropotency. One non-limiting example of such an assay is described inExample 74 herein.

The term half maximal effective concentration (EC₅₀) generally refers tothe concentration which induces a response halfway between the baselineand maximum, by reference to the dose response curve. EC₅₀ is used as ameasure of the potency of a compound and represents the concentrationwhere 50% of its maximal effect is observed.

The in vitro potency of the peptides of the invention may be determinedas described above, and the EC₅₀ of the peptide in question determined.The lower the EC₅₀ value, the better the potency.

Biological Activity—In Vivo Pharmacology

In another embodiment the derivatives or peptides of the invention (oranalogues thereof), are potent in vivo, which may be determined as isknown in the art in any suitable animal model, as well as in clinicaltrials.

The diet-induced obese (DIO) mouse is one example of a suitable animalmodel and the effect on body weight, food intake and glucose tolerancecan be assessed during subchronic dosing in this model. Effect on bodyweight and blood glucose may be determined in such mice in vivo, e.g. asdescribed in Example 77 herein. Food intake can be assessed by singlehousing animals and weighing food consumed per day. This model can alsobe used to evaluate effects on glucose tolerance by performing an oralor i.p. glucose tolerance test (OGTT or IPGTT). These tests areperformed by administration of a glucose load orally or i.p. tosemi-fasted animals and subsequent blood glucose measure for up to threehours.

Pharmacokinetics Profile

According to the third functional embodiment, the peptides of theinvention have improved pharmacokinetic properties such as increasedterminal half-life.

The pharmacokinetic properties of the peptides of the invention maysuitably be determined in-vivo in pharmacokinetic (PK) studies. Suchstudies are conducted to evaluate how pharmaceutical compounds areabsorbed, distributed, and eliminated in the body, and how theseprocesses affect the concentration of the compound in the body, over thecourse of time.

In the discovery and preclinical phase of pharmaceutical drugdevelopment, animal models such as the mouse, rat, monkey, dog, or pig,may be used to perform this characterisation. Any of these models can beused to test the pharmacokinetic properties of the peptides of theinvention.

In such studies, animals are typically administered with a single doseof the drug, either intravenously (i.v.), subcutaneously (s.c.), ororally (p.o.) in a relevant formulation. Blood samples are drawn atpredefined time points after dosing, and samples are analysed forconcentration of drug with a relevant quantitative assay. Based on thesemeasurements, time-plasma concentration profiles for the compound ofstudy are plotted and a so-called non-compartmental pharmacokineticanalysis of the data is performed.

For most compounds, the terminal part of the plasma-concentrationprofiles will be linear when drawn in a semi-logarithmic plot,reflecting that after the initial absorption and distribution, drug isremoved from the body at a constant fractional rate. The rate (lambda Zor λ_(z)) is equal to minus the slope of the terminal part of the plot.From this rate, also a terminal half-life may be calculated, asT_(1/2)=ln(2)/λ_(z) (see e.g. Johan Gabrielsson and Daniel Weiner:Pharmacokinetics and Pharmacodynamic Data Analysis. Concepts &Applications, 3rd Ed., Swedish Pharmaceutical Press, Stockholm, 2000).

Clearance can be determined after i.v. administration and is defined asthe dose (D) divided by area under the curve (AUC) on the plasmaconcentration versus time profile (Rowland, M and Tozer T N: ClinicalPharmacokinetics: Concepts and Applications, 3^(rd) edition, 1995Williams Wilkins).

The estimate of terminal half-life and/or clearance is relevant forevaluation of dosing regimens and an important parameter in drugdevelopment, in the evaluation of new drug compounds.

Pharmacokinetics Profile—Half Life In Vivo in Rats

According to the third functional embodiment, the peptides of theinvention have improved pharmacokinetic properties compared to hGLP-1 orhglucagon. Preferably the peptides of the invention have pharmacokineticproperties suitable for once daily administration or less.

In one embodiment the pharmacokinetic properties may be determined asterminal half-life (T_(1/2)) in vivo in rats after i.v. and s.c.administration. In additional embodiments, the terminal half-life is atleast 1 hour, preferably at least 3 hours, preferably at least 4 hours,even more preferably at least 5 hours, or most preferably at least 6hours.

Pharmacokinetics Profile—Half Life In Vivo in Mice

According to the third functional embodiment, the peptides of theinvention have improved pharmacokinetic properties compared to hGLP-1 orhglucagon. Preferably the peptides of the invention have pharmacokineticproperties suitable for once daily administration or less.

In one embodiment the pharmacokinetic properties may be determined asterminal half-life (T_(1/2)) in vivo in mice after i.v. and s.c.administration. In additional embodiments, the terminal half-life is atleast 1 hour, preferably at least 3 hours, preferably at least 4 hours,even more preferably at least 5 hours, or most preferably at least 6hours. A suitable assay for determining terminal half-life in mice afters.c. administration is disclosed in Example 78 herein.

Pharmacokinetics Profile—Half Life In Vivo in Minipigs

According to the third functional embodiment, the peptides of theinvention have improved pharmacokinetic properties compared to hGLP-1(i.e. human GLP-1) or hglucagon (i.e. human glucagon) and preferablysuitable for once daily or once weekly administration. In one embodimentthe pharmacokinetic properties may be determined as terminal half-life(T_(1/2)) in vivo in minipigs after i.v. administration, e.g. asdescribed below.

In one embodiments, the terminal half-life in minipigs is at least 5hours, preferably at least 10 hours, even more preferably at least 15hours, or most preferably at least 20 hours.

The half-life in vivo in minipigs of the glucagon derivative may bedetermined according to the following method:

The purpose of this study is to determine the pharmacokinetic propertiesin vivo of the glucagon derivatives after i.v. administration tominipigs. This is done in a pharmacokinetic (PK) study, where amongother parameters the terminal half-life and the clearance of thederivative in question is determined. Increasing the terminal half-lifeand decreasing the clearance means that the compound of study iseliminated slower from the body. For glucagon derivatives or analoguesthis entails an extended duration of pharmacological effect.

Female Göttingen minipigs are obtained from Ellegaard Göttingen Minipigs(Dalmose, Denmark) approximately 7-14 months of age and weighing fromapproximately 16-35 kg are used in the studies. The minipigs are housedeither individually (pigs with permanent catheters) or in a group, andare fed restrictedly once or twice daily with SDS minipig diet (SpecialDiets Services, Essex, UK).

In some studies two permanent central venous catheters are implanted invena cava caudalis or cranialis in each animal after at least 2 weeks ofacclimatiation. The animals are allowed 1 week recovery after thesurgery, and are then used for repeated pharmacokinetic studies with asuitable wash-out period between successive glucagon derivative dosings.In other studies the animals are acclimatized for 1 week, after whichthey are used for repeated pharmacokinetic studies with a suitablewash-out period between successive glucagon derivative dosings. On eachdosing occasion these pigs are instrumented with a venflon in one earvein through which the derivatives were dosed. Blood sampling areperformed by venipuncture in v. jugularis or v. cava cranialis

The animals are either unfasted or fasted for approximately 18 h beforedosing and from 0 to 4 h after dosing, but have ad libitum access towater during the whole period.

The glucagon derivatives are usually dissolved in 50 mM sodiumphosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4 to aconcentration of usually from 20-60 nmol/ml. Intravenous injections (thevolume corresponding to usually 2-3 nmol/kg, for example 0.1 ml/kg) ofthe compounds are given through one catheter or through the venflon, andblood are sampled at predefined time points for up till 13 days postdosing (preferably through the other catheter or by venipuncture). Bloodsamples (for example 0.8 ml) are collected in tubes with EDTA buffer (8mM) (sometimes aprotinin 500 KIU/ml blood was added) and thencentrifuged at 4° C. and 1942 G for 10 minutes. Plasma is pippetted intoMicronic tubes on dry ice, and kept at −20° C. until analyzed for plasmaconcentration of the respective glucagon derivative using an appropriatequantitative assay like ELISA or LC-MS.

Based on these measurements, time-plasma concentration profiles for thecompound of study are plotted and a so-called non-compartmentalpharmacokinetic analysis of the data is performed in WinNonlin v. 5.0 orPhoenix v. 6.2 (Pharsight Inc., Mountain View, Calif., USA) or otherrelevant software for PK analysis. For most compounds, the terminal partof the plasma-concentration profiles will be linear when drawn in asemi-logarithmic plot, reflecting that after the initial distribution,drug is removed from the body at a constant fractional rate. The rate(lambda Z or λz) is equal to minus the slope of the terminal part of theplot. From this rate, also the terminal half-life may be calculated, asT½=ln(2)/λz (see e.g. Johan Gabrielsson and Daniel Weiner:Pharmacokinetics and Pharmacodynamic Data Analysis. Concepts &Applications, 3rd Ed., Swedish Pharmaceutical Press, Stockholm (2000)).Clearance is defined as the dose (D) divided by area under the curve(AUC) on the plasmaconcentration versus time profile (Rowland, M andTozer T N: Clinical Pharmacokinetics: Concepts and Applications, 3rdedition, 1995 Williams Wilkins).

Physicochemical Properties

According to the fourth embodiment, the peptides of the invention haveadvantageous physicochemical properties. These properties include butare not limited to physical stability, chemical stability and/orsolubility. These and other physicochemical properties may be measuredusing standard methods known in the art of protein chemistry. In oneembodiment these properties are improved as compared to native glucagon(SEQ ID NO:1). More pronounced self-association of the peptidesself-association properties of the peptides may be at least partlyresponsible for the improved physical stability and/or solubility.

Non-limiting examples of assays to investigate biophysical propertiesare described in Example 76 and Example 79 herein.

In one embodiment the glucagon derivative of the invention has more than70% recovery in the ThT fibrillation assay. In one embodiment theglucagon derivative of the invention has more than 90% recovery in theThT fibrillation assay. In one embodiment the glucagon derivative of theinvention has about 100% recovery in the ThT fibrillation assay. In oneembodiment the glucagon derivative of the invention has more than 7hours lag time in the ThT fibrillation assay. In one embodiment theglucagon derivative of the invention has more than 20 hours lag time inthe ThT fibrillation assay. In one embodiment the glucagon derivative ofthe invention has 45 hours lag time or more in the ThT fibrillationassay. A glucagon derivative as described herein, wherein said ThTfibrillation assay is as described in Example 76 herein.

In one embodiment the glucagon derivative of the invention has less than14% degradation in the chemical stability assay. In one embodiment theglucagon derivative of the invention has less than 13% degradation inthe chemical stability assay. In one embodiment the glucagon derivativeof the invention has less than 12% degradation in the chemical stabilityassay. In one embodiment the glucagon derivative of the invention hasless than 10% degradation in the chemical stability assay. In oneembodiment the glucagon derivative of the invention has less than 9%degradation in the chemical stability assay. In one embodiment theglucagon derivative of the invention has less than 7% degradation in thechemical stability assay. In one embodiment the glucagon derivative ofthe invention has less than 5% degradation in the chemical stabilityassay. In one embodiment the glucagon derivative of the invention hasless than 3% degradation in the chemical stability assay. In oneembodiment the glucagon derivative of the invention has less than 2%degradation in the chemical stability assay. In one embodiment thechemical stability assay is as described in Example 79 herein.

Stability

The term “physical stability” of the GLP-1 receptor agonist peptidepreparation as used herein refers to the tendency of the protein to formbiologically inactive and/or insoluble aggregates of the protein as aresult of exposure of the protein to thermo-mechanical stresses and/orinteraction with interfaces and surfaces that are destabilizing, such ashydrophobic surfaces and interfaces. Physical stability of the aqueousprotein preparations is evaluated by means of visual inspection and/orturbidity measurements after exposing the preparation filled in suitablecontainers (e.g. cartridges or vials) to mechanical/physical stress(e.g. agitation) at different temperatures for various time periods.Visual inspection of the preparations is performed in a sharp focusedlight with a dark background. The turbidity of the preparation ischaracterized by a visual score ranking the degree of turbidity forinstance on a scale from 0 to 3 (a preparation showing no turbiditycorresponds to a visual score 0, and a preparation showing visualturbidity in daylight corresponds to visual score 3). A preparation isclassified physically unstable with respect to protein aggregation, whenit shows visual turbidity in daylight. Alternatively, the turbidity ofthe preparation can be evaluated by simple turbidity measurementswell-known to the skilled person. Physical stability of the aqueousprotein preparations can also be evaluated by using a spectroscopicagent or probe of the conformational status of the protein. The probe ispreferably a small molecule that preferentially binds to a non-nativeconformer of the protein. One example of a small molecular spectroscopicprobe of protein structure is Thioflavin T. Thioflavin T is afluorescent dye that has been widely used for the detection of amyloidfibrils. In the presence of fibrils, and perhaps other proteinconfigurations as well, Thioflavin T gives rise to a new excitationmaximum at about 450 nm and enhanced emission at about 482 nm when boundto a fibril protein form. Unbound Thioflavin T is essentiallynon-fluorescent at the wavelengths.

The term “chemical stability” of the protein preparation as used hereinrefers to changes in the covalent protein structure leading to formationof chemical degradation products with potential less biological potencyand/or potential increased immunogenic properties compared to the nativeprotein structure. Various chemical degradation products can be formeddepending on the type and nature of the native protein and theenvironment to which the protein is exposed. Increasing amounts ofchemical degradation products is often seen during storage and use ofthe protein preparation. Most proteins are prone to deamidation, aprocess in which the side chain amide group in glutaminyl or asparaginylresidues is hydrolysed to form a free carboxylic acid or asparaginylresidues to form an IsoAsp derivative. Other degradations pathwaysinvolves formation of high molecular weight products where two or moreprotein molecules are covalently bound to each other throughtransamidation and/or disulfide interactions leading to formation ofcovalently bound dimer, oligomer and polymer degradation products(Stability of Protein Pharmaceuticals, Ahern. T. J. & Manning M. C.,Plenum Press, New York 1992). Oxidation (of for instance methionineresidues) can be mentioned as another variant of chemical degradation.The chemical stability of the protein preparation can be evaluated bymeasuring the amount of the chemical degradation products at varioustime-points after exposure to different environmental conditions (theformation of degradation products can often be accelerated by forinstance increasing temperature). The amount of each individualdegradation product is often determined by separation of the degradationproducts depending on molecule size and/or charge using variouschromatography techniques (e.g. SEC-HPLC and/or RP-HPLC). Since HMWPproducts are potentially immunogenic and not biologically active, lowlevels of HMWP are advantageous.

The term “stabilized preparation” refers to a preparation with increasedphysical stability, increased chemical stability or increased physicaland chemical stability. In general, a preparation must be stable duringuse and storage (in compliance with recommended use and storageconditions) until the expiration date is reached.

Solubility

In the present context, if not stated otherwise, the terms “soluble”,“solubility”, “soluble in aqueous solution”, “aqueous solubility”,“water soluble”, “water-soluble”, “water solubility” and“water-solubility”, refer to the solubility of a compound in water or inan aqueous salt or aqueous buffer solution, for example a 10 mMphosphate solution, or in an aqueous solution containing othercompounds. Solubility may be assessed using the following assay.

pH Dependent Solubility Assay

The solubility of peptides and proteins depends on the pH of thesolution. Often a protein or peptide precipitates at or close to itsisoelectric point (pl), at which its net charge is zero. At low pH (i.e.lower than the pl) proteins and peptides are typically positivelycharged, at pH higher than the pl they are negatively charged.

It is advantageous for a therapeutic peptide if it is soluble in asufficient concentration at a given pH, which is suitable for bothformulating a stable drug product and for administrating the drugproduct to the patient e.g. by subcutaneous injection.

Solubility versus pH curves are measured as described: a formulation ora peptide solution in water is prepared and aliquots are adjusted to pHvalues in the desired range by adding HCl and NaOH. These samples areleft equilibrating at room temperature for 2-4 days. Then the samplesare centrifuged. A small aliquot of each sample is withdrawn for reverseHPLC analysis for determination of the concentration of the proteins insolution. The pH of each sample is measured after the centrifugation,and the concentration of each protein is depicted versus the measuredpH.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

The compounds of the invention may be a soluble glucagon/GLP-1 receptorco-agonist, for example with solubility of at least 0.1 mmol/l, 0.2mmol/l, at least 0.5 mmol/l, at least 2 mmol/l, at least 4 mmol/l, atleast 8 mmol/l, at least 10 mmol/l, or at least 15 mmol/l.

DPP-IV Stability

In one embodiment the glucagon derivative of the invention is DPP-IVprotected. The term “DPP-IV protected” as used herein referring to apolypeptide means a polypeptide which has been chemically modified inorder to render said compound resistant to the plasma peptidasedipeptidyl aminopeptidase-4 (DPP-IV). The DPP-IV enzyme in plasma isknown to be involved in the degradation of several peptide hormones,e.g. glucagon, GLP-1, GLP-2, oxyntomodulin etc. Thus, a considerableeffort is being made to develop analogues and derivatives of thepolypeptides susceptible to DPP-IV mediated hydrolysis in order toreduce the rate of degradation by DPP-IV. In one embodiment the glucagonderivative is a DPPIV protected compound. In one embodiment the glucagonderivative is DPPIV stabilised.

In one embodiment the compounds of the invention may be stabilizedagainst DPP-IV cleavage in an albumin free assay:

10 μM of peptide is incubated with DPP-IV (2 μg/ml) in duplicate at 37°C. in a HEPES buffer to which 0.005% Tween20 is added. In the experimenthuman GLP-1 is used as a positive control. Aliqouts of sample are takenat 3, 15, 30, 60, 120 and 240 min and three volumes of ethanol are addedto stop the reaction. The samples are analysed by LC-MS for parentpeptide. Data are plotted according to 1^(st) kinetics and the stabilityis reported as half-lives.

COMBINATIONS

In one embodiment the invention relates to the glucagon derivative asdescribed herein in combination with a GLP-1 compound or with an insulincompound. In one embodiment the invention relates to the glucagonderivative as described herein in combination with a GLP-1 compound. Inone embodiment the invention relates to the glucagon derivative asdescribed herein in combination with an insulin compound.

In one embodiment the GLP-1 compound of the combination isN-epsilon26-((S)-4-Carboxy-4-hexadecanoylamino-butyryl)[Arg34]GLP-1-(7-37):

(Compound G1); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof. In one embodiment the GLP-1 compound of the combinationisN-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg126Arg34,Lys37]GLP-1-(7-37):

(Compound G2); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof. In one embodiment the GLP-1 compound of the combinationisN-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37):

(Compound G3); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof. In one embodiment the GLP-1 compound of the combinationisN-epsilon-37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,22,35,Lys37]GLP-1-(7-37):

(Compound G4); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof.

In one embodiment the insulin compound of the combination isN-epsilon-B29-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyryl]desB30 human insulin

PHARMACEUTICAL COMPOSITIONS

In one embodiment the invention relates to a pharmaceutical compositioncomprising the derivative of the invention and one or morepharmaceutically acceptable excipients. In one embodiment thecomposition is suited for parenteral administration, such as SC, IM orIV administration. The terms “pharmaceutical composition” and“composition” are used interchangeably herein.

Pharmaceutical compositions containing a derivative of the invention maybe prepared by conventional techniques, e.g. as described in Remington'sPharmaceutical Sciences, 1985 or in Remington: The Science and Practiceof Pharmacy, 19th edition, 1995.

In one embodiment the pharmaceutical composition in unit dosage formcomprises from about 0.01 mg to about 1000 mg, such as from about 0.1 mgto about 500 mg, from about 0.5 mg to about 5 mg, e.g. from about 0.5 mgto about 200 mg, of a glucagon derivative as described herein. In oneembodiment the invention relates to a pharmaceutical compositioncomprising a derivative of the invention, wherein said derivative ispresent in a concentration from about 0.01 mg/mL to about 25 mg/mL, suchas from about 0.05 mg/mL to about 5 mg/mL and from about 0.1 mg/mL toabout 2 mg/mL, and wherein said composition has a pH from 2.0 to 10.0.The pharmaceutical composition may comprise a derivative of theinvention, wherein said derivative is present in a concentration fromabout 0.01 mg/mL to about 50 mg/mL, and wherein said composition has apH from 2.0 to 10.0.

In one embodiment the pharmaceutical composition comprises an aqueoussolution of a derivative of the invention, and a buffer, wherein saidderivative is present in a concentration from 0.01 mg/mL or above, andwherein said composition has a pH from about 2.0 to about 10.0. Inanother embodiment the pharmaceutical composition comprises an aqueoussolution of a derivative of the invention, and a buffer, wherein saidderivative is present in a concentration from 0.01 mg/mL or above, andwherein said composition has a pH from about 6.5 to about 8.5.

In one embodiment the composition of the invention has a pH from about2.0 to about 10.0. In another embodiment the composition has a pH fromabout 6.5 to about 8.5. In a further embodiment the composition has a pHfrom about 7.0 to about 8.5, such as from about 7.2 to about 8.2.

The composition may further comprise a buffer system, preservative(s),isotonicity agent(s), chelating agent(s), stabilizers and surfactants.In one embodiment the pharmaceutical composition is an aqueouscomposition, i.e. a composition comprising water. Such composition istypically a solution or a suspension. In a further embodiment of theinvention the pharmaceutical composition is an aqueous solution. Theterm “aqueous composition” is defined as a composition comprising atleast 50% w/w water. Likewise, the term “aqueous solution” is defined asa solution comprising at least 50% w/w water. In one embodiment thecomposition comprises a non-aqeuous organic solvent.

In another embodiment the pharmaceutical composition is a freeze-driedcomposition to which solvents and/or diluents are added prior to use,e.g. by the physician or the patient.

In another embodiment the pharmaceutical composition is a driedcomposition (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In one embodiment the invention relates to a composition comprising thederivative of the invention and one or more other active ingredients,such as GLP-1, insulin or analogues and/or derivatives thereof. In oneembodiment the invention relates to a composition comprising thederivative of the invention and GLP-1 or analogues and/or derivativesthereof. In one embodiment the invention relates to a compositioncomprising the derivative of the invention and insulin or analoguesand/or derivatives thereof. A composition comprising the derivative ofthe invention and one or more other active ingredients may be referredto as a “co-formulation”. In one embodiment such co-formulations arephysically stable and/or chemically stable compositions.

The fact that the derivatives of the invention may be soluble at neutralpH, may allow a co-formulation with insulin and allow for more stableblood glucose levels and a reduced number of hypoglycaemic episodes, aswell as a reduced risk of diabetes related complications.

In one embodiment the pharmaceutical composition further comprises oneor more additional therapeutically active compounds or substances. Inone embodiment the additional therapeutically active compound is a GLP-1compound or an insulin compound. In one embodiment the additionaltherapeutically active compound is a GLP-1 compound. In one embodimentthe additional therapeutically active compound is an insulin compound.In one embodiment the additional therapeutically active compound is theGLP-1 compoundN-epsilon26-((S)-4-Carboxy-4-hexadecanoylamino-butyryl)[Arg34]GLP-1-(7-37):

(Compound G1); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof. In one embodiment the additional therapeutically activecompound is the GLP-1 compoundN-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg126,Arg34,Lys37]GLP-1-(7-37):

(Compound G2); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof. In one embodiment the additional therapeutically activecompound is the GLP-1 compoundN-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37):

(Compound G3); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof. In one embodiment the additional therapeutically activecompound is the GLP-1 compoundN-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,22,35,Lys37]GLP-1-(7-37):

(Compound G4); or a pharmaceutically acceptable salt, amide, alkyl, orester thereof.

In one embodiment the additional therapeutically active compound is theinsulin compoundN-epsilon-B29-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyryl]desB30human insulin

In one embodiment the additional therapeutically active compound is apharmaceutically acceptable salt, amide, alkyl, or ester of Compound G5.

PHARMACEUTICAL ADMINISTRATION

The derivative of the invention may be administered parenterally to apatient. The route of administration of the derivative may beintramuscular (IM), subcutaneous (SC), or intravenous (IV). It isrecommended that the dosage of the compositions comprising thederivative of this invention which is to be administered to the patientbe selected by a physician.

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. In one embodiment thecompositions comprising the derivative of the invention can be used inready to use pen devices for glucagon administration. Alternatively,parenteral administration can be performed by means of an infusion pump.In one embodiment the compositions comprising the derivative of theinvention can be used in pumps for glucagon administration. Parenteraladministration may be nasal administration. As a further option, theglucagon preparations containing the derivative of the invention canalso be adapted to transdermal administration, e.g. by needle-freeinjection or from a patch, optionally an iontophoretic patch, ortransmucosal, e.g. buccal, administration.

A typical dosage of a derivative or composition of the invention whenemployed in a method according to the invention is in the range of fromabout 0.0001 to about 1 mg/kg body weight per day, preferably from about0.001 to about 1 mg/kg body weight, more preferably from about 0.005 toabout 0.02 mg/kg body. As described above, derivatives of the inventionmay be administered or applied in combination with one or moreadditional therapeutically active compounds or substances, and suitableadditional compounds or substances may be selected, for example, fromantidiabetic agents, antihyperlipidemic agents, antiobesity agents,antihypertensive agents and agents for the treatment of complicationsresulting from, or associated with, diabetes.

Suitable antidiabetic agents include insulin, insulin derivatives oranalogues, GLP-1 (glucagon like peptide-1) derivatives or analogues[such as those disclosed in WO 98/08871 (Novo Nordisk A/S), or otherGLP-1 analogues such as exenatide (Byetta, Eli Lilly/Amylin; AVE0010,Sanofi-Aventis), taspoglutide (Roche), albiglutide (Syncria,GlaxoSmithKline)], amylin, amylin analogues (e.g. Symlin/Pramlintide) aswell as orally active hypoglycemic agents.

PHARMACEUTICAL INDICATIONS

The invention also relates to a derivative of the invention, for use inmedicine. In one embodiment the invention relates to the glucagonderivative, optionally in combination with one or more additionaltherapeutically active compounds, for use in therapy. In one embodimentthe terms “glucagon derivative” and “derivative” are usedinterchangeably herein in connection with a medical use and refers tothe glucagon derivative of the invention. In one embodiment the terms“medicine” and “therapy” are used interchangeably herein. In oneembodiment the invention relates to a method of treatment or preventionof a disease or disorder, e.g. one or more of the diseases or disordersmentioned herein in relation to specific medical uses. In one embodimentthe invention relates to a method for the preparation of a medicamentfor use in therapy, e.g. in the specific medical uses defined herein.

In one embodiment the invention relates to a glucagon derivative of theinvention, optionally in combination with one or more additionaltherapeutically active compounds, for use in medicine.

In one embodiment the derivative is used for treating and/or preventinghypoglycemia. In one embodiment the invention relates to a method fortreating and/or preventing hypoglycemia comprising administering atherapeutically effective amount of the derivative of the invention to apatient in need thereof.

In one embodiment the invention relates to the glucagon derivative foruse in treating or preventing type 2 diabetes. In one embodiment theglucagon derivative of the invention is for use in treating impairedglucose tolerance. In one embodiment the invention relates to theglucagon derivative for use in delaying or preventing diseaseprogression in type 2 diabetes. In one embodiment the invention relatesto a glucagon derivative of the invention, optionally in combinationwith one or more additional therapeutically active compounds, for use indelaying the progression from type 2 diabetes to insulin-requiringdiabetes, for use regulating appetite, for use in preventing weightregain after successful weight loss, for use in inducing satiety, foruse in preventing weight regain after successful weight loss, for use intreating a disease or state related to overweight or obesity. In oneembodiment the invention relates to the glucagon derivative for use indelaying the progression from impaired glucose tolerance (IGT) to type 2diabetes. In one embodiment the invention relates to the glucagonderivative for use in delaying the progression from type 2 diabetes toinsulin-requiring diabetes.

In one embodiment the invention relates to a method for treating orpreventing hypoglycemia, type 2 diabetes, impaired glucose tolerance,type 1 diabetes and obesity, comprising administering to a patient inneed thereof, an effective amount of a glucagon derivative according tothe invention, optionally in combination with one or more additionaltherapeutically active compounds. In one embodiment the inventionrelates to the glucagon derivative for use in treating or preventinghypoglycemia, type 2 diabetes, impaired glucose tolerance, type 1diabetes and obesity.

In one embodiment the invention relates to the glucagon derivative foruse in treating obesity or preventing overweight. In one embodiment theinvention relates to a method for treating obesity or preventingoverweight, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative according to the invention,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in reducingbody weight, for use in regulating appetite, for use in increasingenergy expenditure, for use in inducing satiety, for use in preventingweight regain after successful weight loss, for use in treating adisease or state related to overweight or obesity, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to the invention, optionally incombination with one or more additional therapeutically activecompounds. In one embodiment the invention relates to the glucagonderivative for use in decreasing food intake. In one embodiment theinvention relates to the glucagon derivative for use in increasingenergy expenditure. In one embodiment the invention relates to theglucagon derivative for use in reducing body weight. In one embodimentthe invention relates to the glucagon derivative for use in regulatingappetite. In one embodiment the invention relates to the glucagonderivative for use in inducing satiety. In one embodiment the inventionrelates to the glucagon derivative for use in preventing weight regainafter successful weight loss. In one embodiment the invention relates tothe glucagon derivative for use in treating a disease or state relatedto overweight or obesity. In one embodiment the invention relates to theglucagon derivative for use in treating bulimia. In one embodiment theinvention relates to the glucagon derivative for use in treatingbinge-eating.

In one embodiment the invention relates to use of a glucagon derivativeaccording to the invention, for the preparation of a medicament.

In one embodiment the invention relates to use of a glucagon derivativeaccording to the invention, for the preparation of a medicament for thetreatment or prevention of hyperglycemia, type 2 diabetes, impairedglucose tolerance, type 1 diabetes and obesity. In one embodiment theglucagon derivative of the invention is for use in the treatment orprevention of hyperglycemia, type 2 diabetes, impaired glucosetolerance, type 1 diabetes and obesity.

In one embodiment the glucagon derivative of the invention is for use inthe treatment or prevention of atherosclerosis. In one embodiment theglucagon derivative of the invention is for use in treatinghypertension. In one embodiment the glucagon derivative of the inventionis for use in treating dyslipidemia. In one embodiment the glucagonderivative of the invention is for use in treating coronary heartdisease. In one embodiment the glucagon derivative of the invention isfor use in treating hepatic steatosis.

In one embodiment the invention relates to a method for decreasing foodintake, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use inincreasing energy expenditure, comprising administering to a patient inneed thereof, an effective amount of a glucagon derivative as describedherein, optionally in combination with one or more additionaltherapeutically active compounds.

In one embodiment the invention relates to a method for use in reducingbody weight, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in delayingthe progression from impaired glucose tolerance (IGT) to type 2diabetes, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in delayingthe progression from type 2 diabetes to insulin-requiring diabetes,comprising administering to a patient in need thereof, an effectiveamount of a glucagon derivative as described herein, optionally incombination with one or more additional therapeutically activecompounds.

In one embodiment the invention relates to a method for use inregulating appetite, comprising administering to a patient in needthereof, an effective amount of a glucagon derivative as describedherein, optionally in combination with one or more additionaltherapeutically active compounds.

In one embodiment the invention relates to a method for use in inducingsatiety, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use inpreventing weight regain after successful weight loss, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative as described herein, optionally in combination withone or more additional therapeutically active compounds.

In one embodiment the invention relates to a method for use in treatinga disease or state related to overweight or obesity, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative as described herein, optionally in combination withone or more additional therapeutically active compounds.

In one embodiment the invention relates to a method for use in treatingbulimia, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in treatingbinge-eating, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in treatingatherosclerosis, comprising administering to a patient in need thereof,an effective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in treatinghypertension, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in treatingtype 2 diabetes, comprising administering to a patient in need thereof,an effective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in treatingimpaired glucose tolerance, comprising administering to a patient inneed thereof, an effective amount of a glucagon derivative as describedherein, optionally in combination with one or more additionaltherapeutically active compounds.

In one embodiment the invention relates to a method for use in treatingdyslipidemia, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative as described herein,optionally in combination with one or more additional therapeuticallyactive compounds.

In one embodiment the invention relates to a method for use in treatingcoronary heart disease, comprising administering to a patient in needthereof, an effective amount of a glucagon derivative as describedherein, optionally in combination with one or more additionaltherapeutically active compounds.

In one embodiment the invention relates to a method for use in treatinghepatic steatosis, comprising administering to a patient in needthereof, an effective amount of a glucagon derivative as describedherein, optionally in combination with one or more additionaltherapeutically active compounds.

In one embodiment the invention relates to use of a glucagon derivativeas described herein, for the preparation of a medicament for delaying orpreventing disease progression in type 2 diabetes, treating obesity orpreventing overweight, for decreasing food intake, increase energyexpenditure, reducing body weight, delaying the progression fromimpaired glucose tolerance (IGT) to type 2 diabetes; delaying theprogression from type 2 diabetes to insulin-requiring diabetes;regulating appetite; inducing satiety; preventing weight regain aftersuccessful weight loss; treating a disease or state related tooverweight or obesity; treating bulimia; treating binge-eating; treatingatherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia,coronary heart disease, hepatic steatosis, treatment of beta-blockerpoisoning, use for inhibition of the motility of the gastrointestinaltract, useful in connection with investigations of the gastrointestinaltract using techniques such as x-ray, CT- and NMR-scanning.

In one embodiment the invention relates to novel stable and protractedGLP-1/glucagon receptor co-agonists, to the use of said peptides intherapy, to methods of treatment comprising administration of saidpeptides to patients, and to the use of said peptides in the manufactureof medicaments for use in the treatment of diabetes, obesity and relateddiseases and conditions.

In one embodiment the derivative of the invention is used for treatmentor prevention of hypoglycemia, insulin induced hypoglycemia, reactivehypoglycemia, diabetic hypoglycemia, non-diabetic hypoglycemia, fastinghypoglycemia, drug-induced hypoglycemia, gastric by-pass inducedhypoglycemia, hypoglycemia in pregnancy, alcohol induced hypoglycemia,insulinoma and/or Von Girkes disease.

In one embodiment the derivatives of the invention are for use ininhibition of the motility of the gastrointestinal tract, which isuseful in connection with investigations of the gastrointestinal tractusing techniques such as x-ray, CT- and NMR-scanning.

In one embodiment the derivative is for use in treatment of beta-blockerpoisoning. In one embodiment the derivative is for use in treatment orprevention of hepatic steatosis. In one embodiment the derivative is foruse in treatment or prevention of hypoglycaemia. In one embodiment thederivative is for use in treatment or prevention of insulin inducedhypoglycaemia. In one embodiment the derivative is for use in treatmentor prevention of reactive hypoglycaemia. In one embodiment thederivative is for use in treatment or prevention of diabetichypoglycaemia. In one embodiment the derivative is for use in treatmentor prevention of non-diabetic hypoglycaemia. In one embodiment thederivative is for use in treatment or prevention of fastinghypoglycaemia. In one embodiment the derivative is for use in treatmentor prevention of drug-induced hypoglycaemia. In one embodiment thederivative is for use in treatment or prevention of gastric by-passinduced hypoglycaemia. In one embodiment the derivative is for use intreatment or prevention of hypoglycemia in pregnancy. In one embodimentthe derivative is for use in treatment or prevention of alcohol-inducedhypoglycaemia. In one embodiment the derivative is for use in treatmentor prevention of insulinoma. In one embodiment the derivative is for usein treatment or prevention of Von Girkes disease.

In one embodiment the derivative is administered in a dosage regimewhich provides a therapeutically effective amount of said derivative. Asused herein, the term “therapeutically effective amount” of a derivativeof the invention refers to an amount sufficient to cure, alleviate orpartially arrest the clinical manifestations of a given disease and/orits complications. An amount adequate to accomplish this is defined as a“therapeutically effective amount”. Effective amounts for each purposewill depend on the severity of the disease or injury, as well as on theweight and general state of the patient. It will be understood thatdetermination of an appropriate dosage may be achieved using routineexperimentation, by constructing a matrix of values and testingdifferent points in the matrix, all of which is within the level ofordinary skill of a trained physician or veterinarian.

The terms “treatment”, “treating” and other variants thereof as usedherein refer to the management and care of a patient or patient for thepurpose of combating a condition, such as a disease or a disorder. Theterms are intended to include the full spectrum of treatments for agiven condition from which the patient is suffering, such asadministration of the active compound(s) in question to alleviatesymptoms or complications thereof, to delay the progression of thedisease, disorder or condition, to cure or eliminate the disease,disorder or condition, and/or to prevent the condition, in thatprevention is to be understood as the management and care of a patientfor the purpose of combating the disease, condition, or disorder, andincludes the administration of the active compound(s) in question toprevent the onset of symptoms or complications. The patient to betreated is preferably a mammal, in particular a human being, buttreatment of other animals, such as dogs, cats, cows, horses, sheep,goats or pigs, is within the scope of the invention.

INTERMEDIATE PRODUCTS

In one embodiment the invention relates to an intermediate product inthe form of a glucagon peptide which comprises a C-terminal amide andany one of the following modifications as compared to glucagon (SEQ IDNO: 1):

a) [Aib2,Leu10,Lys6,Arg20,Leu27,Ser28];

b) [Aib2,Leu10,Arg20,Lys21,Leu27,Ser28];

c) [Aib2,Leu10,Arg20,Lys24,Leu27,Ser28];

d) [Aib2,Leu10,Arg20,Leu27,Lys28];

e) [Aib2,Leu10,Arg20,Leu27,Ser28,Lys29];

f) [Aib2,Leu10,Arg20,Leu27,Ser28];

g) [Aib2,Leu10,Lys16,Lys17,Glu21,Leu27];

h) [Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29];

i) [Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29];

j) [Aib2,Leu10,Lys16,Arg20,Glu21,Leu27];

k) [Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29];

1) [Aib2,Leu10,Arg20,Glu21,Leu27,Lys29];

m) Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29];

n) [Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29];

o) [Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29];

p) [Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28];

q) [Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27];

r) [Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27];

s) [Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27];

t) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

u) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29];

v) [Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

w) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

x) [Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28];

y) [Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

z) [Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28];

aa) [Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

bb) [Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29];

cc) [Aib2,Leu10,Lys16,Glu21,Leu27];

dd) [Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

ee) [Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28];

ff) [Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28];

gg) [Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

hh) [Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

ii) [Acb2,His3,Leu10,Glu15,Leu27,Lys28];

jj) [Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]

kk) [Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28];

ll) [Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29];

mm) [Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29];

nn) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29];

oo) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

pp) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

qq) [Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28];

rr) [Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

ss) [Acb2,Leu10,Leu16,Arg20,Leu27,Lys28];

tt) [Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28];

uu) [Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28];

w) [Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28];

ww) [Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28];

xx) [Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

yy) [Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28];

zz) [Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

aaa) [Aib2,Leu10,Leu16,Leu27,Lys28];

bbb) [Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

ccc) [Aib2,Leu10,Leu16,Arg20,Leu27,Lys28];

ddd) [Aib2,Leu10,Arg20,Glu21,Leu27,Lys28];

eee) [Aib2,Leu10,Glu15,Arg20,Leu27,Lys28];

fff) [Aib2,Leu10,Ala16,Arg20,Leu27,Lys28];

ggg) [Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28];

hhh) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

iii) [Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

jjj) [Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28];

kkk) [Acb2,Leu10,Glu15,Leu27,Lys28];

lll) [Aib2,Leu10,Arg12,Ala16,Leu27,Lys28];

mmm) [Aib2,Leu10,Ala16,Arg20,Leu27,Lys29];

nnn) [Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29];

ooo) [Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

ppp) [Aib2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28];

qqq) [Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

rrr) [Aib2,Leu10,Glu15,Lys17,Arg20,Ser21,Leu27,Lys28];

sss) [Aib2,Val10,Ala16,Leu27,Lys28];

ttt) [Aib2,Val10,Leu16,Leu27,Lys28];

uuu) [Aib2,Val10,Arg12,Ala16,Leu27,Lys28]; or

vvv) [Aib2,Val10,Arg12,Leu16,Leu27,Lys28];

or a pharmaceutically acceptable salt, amide, or ester thereof.

In one embodiment the invention relates to an intermediate product inthe form of a glucagon peptide having a C-terminal amide and consistingof one of the modifications a) to ppp) as defined above as compared toglucagon (SEQ ID NO: 1), or a pharmaceutically acceptable salt, amide,or ester thereof.

The glucagon derivatives of the invention may be prepared by thefollowing a stepwise synthesis method comprising (i) preparation of theintermediate glucagon peptide followed by (ii) attachment of thesubstituent. Step (i) of this method can be achieved using standardsolid phase synthesis as described in the experimental section usingprotected amino acids; after cleavage from the resin the glucagonpeptide can be subjected to purification using preparative HPLC asdescribed in the experimental section herein to give the intermediateproduct. Alternatively, step (i) of this method, preparation of theintermediate product, can be carried out using a semirecombinantsynthesis as described in WO2009/083549. Step (ii) of this method, i.e.the attachment of the substituent to the intermediate product leading tothe final product, as well as preparation of the substituent itself canbe achieved using methods described in WO2009/083549.

Embodiments of the Invention

The glucagon derivatives of the invention may further be characterisedby one or more of the following non-limiting embodiments:

1. A glucagon derivative comprising the amino acid sequence of Formula I(corresponding to SEQ ID NO:4 and SEQ ID NO:5):

His-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₇-X₁₈Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀  [I]

wherein

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu, Ile or Val;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, Ala or Lys;

X₂₀ represents Gln, Arg, Glu, Aib or Lys;

X₂₁ represents Asp, Glu, Ser or Lys;

X₂₄ represents Gln, Ala, Arg, Glu, Aib or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys;

X₂₉ represents Thr, Gly, Ser, Gln, Ala, Glu or Lys;

X₃₀ represents Lys, or X₃₀ is absent;

which amino acid sequence comprises a lysine residue at one or more ofpositions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30; and whereinsaid glucagon derivative comprises a substituent comprising a lipophilicmoiety and at least three negatively charged moieties, wherein one ofsaid negatively charged moieties is distal of a lipophilic moiety, isattached at the epsilon position of a lysine residue in one of the aminoacid positions 16, 17, 18, 20, 21, 24, 28, 29, or 30; and wherein saidglucagon derivative is a C-terminal amide, or a pharmaceuticallyacceptable salt or prodrug thereof.

2. A glucagon derivative comprising the amino acid sequence of Formula I(corresponding to SEQ ID NO:2 and SEQ ID NO:3):

His-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₁₇-X₁₈-Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀  [I]

wherein

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu, Ile or Val;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, Ala or Lys;

X₂₀ represents Gln, Arg, Glu, Aib or Lys;

X₂₁ represents Asp, Glu or Lys;

X₂₄ represents Gln, Ala, Arg, Glu, Aib or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys;

X₂₉ represents Thr, Gly, Ser, Gln, Ala, Glu or Lys;

X₃₀ represents Lys, or X₃₀ is absent;

which amino acid sequence comprises a lysine residue at one or more ofpositions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30; and whereinsaid glucagon derivative comprises a substituent comprising a lipophilicmoiety and at least three negatively charged moieties, wherein one ofsaid negatively charged moieties is distal of a lipophilic moiety, isattached at the epsilon position of a lysine residue in one of the aminoacid positions 16, 17, 18, 20, 21, 24, 28, 29, or 30; and wherein saidglucagon derivative is a C-terminal amide, or a pharmaceuticallyacceptable salt or prodrug thereof.

3. A glucagon derivative of Formula I as described above, wherein

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, or Ala;

X₂₀ represents Gln, Arg, Glu, or Lys;

X₂₁ represents Asp, Glu or Lys;

X₂₄ represents Gln, Ala, Arg, or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, or Lys;

X₂₉ represents Thr, Gly, or Lys; and

X₃₀ represents Lys, or X₃₀ is absent.

4. A glucagon derivative of Formula I as described above, wherein asubstituent comprising a lipophilic moiety and three or more negativelycharged moieties, wherein one of said negatively charged moieties isdistal of a lipophilic moiety, and wherein said substituent is attachedat the epsilon position of a Lys in one of the following amino acidpositions of said glucagon derivative: 16, 17, 18, 20, 21, 24, 28, 29,and/or 30.

5. A glucagon derivative of Formula I as described above, wherein

X₂ represents Aib, Acb or Acpr;

X₃ represents Gln or His;

X₁₀ represents Leu;

X₁₂ represents Lys or Arg;

X₁₅ represents Asp or Glu;

X₁₆ represents Ser, Ala, Leu, Thr, Glu, or Lys;

X₁₇ represents Arg or Lys;

X₁₈ represents Arg, or Ala;

X₂₀ represents Gln, Arg, Glu, or Lys;

X₂₁ represents Asp, Glu or Lys;

X₂₄ represents Gln, Ala, Arg, or Lys;

X₂₇ represents Met, Leu or Val;

X₂₈ represents Asn, Ser, or Lys;

X₂₉ represents Thr, Gly, or Lys; and

X₃₀ represents Lys, or X₃₀ is absent.

6. The glucagon derivative of Formula I as described above, wherein X₂represents Aib, Acb or Acpr.

7. The glucagon derivative of Formula I as described above, wherein X₂represents Aib.

8. The glucagon derivative of Formula I as described above, wherein X₂represents Acb.

9. The glucagon derivative of Formula I as described above, wherein X₂represents Acpr.

10. The glucagon derivative of Formula I as described above, wherein X₃represents Gln or His.

11. The glucagon derivative of Formula I as described above, wherein X₃represents Gln.

12. The glucagon derivative of Formula I as described above, wherein X₃represents His.

13. The glucagon derivative of Formula I as described above, wherein X₁₀represents Leu, Ile or Val.

14. The glucagon derivative of Formula I as described above, wherein X₁₀represents Leu.

15. The glucagon derivative of Formula I as described above, wherein X₁₀is Ile.

16. The glucagon derivative of Formula I as described above, wherein X₁₀represents Val.

17. The glucagon derivative of Formula I as described above, wherein X₁₂represents Lys or Arg.

18. The glucagon derivative of Formula I as described above, wherein X₁₂represents Lys.

19. The glucagon derivative of Formula I as described above, wherein X₁₂represents Arg.

20. The glucagon derivative of Formula I as described above, wherein X₁₅represents Asp or Glu.

21. The glucagon derivative of Formula I as described above, wherein X₁₅represents Asp.

22. The glucagon derivative according to any one of the previousembodiments, wherein X₁₅ represents Glu.

23. The glucagon derivative of Formula I as described above, wherein X₁₆represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys.

24. The glucagon derivative of Formula I as described above, wherein X₁₆represents Ser, Ala, Leu, Thr, Glu, Aib, or Lys.

25. The glucagon derivative of Formula I as described above, wherein X₁₆represents Ser, Ala, Leu, Thr, Glu or Lys.

26. The glucagon derivative of Formula I as described above, wherein X₁₆represents Ala, Leu, Thr, Glu or Lys.

27. The glucagon derivative of Formula I as described above, wherein X₁₆represents Ser.

28. The glucagon derivative of Formula I as described above, wherein X₁₆represents Ala.

29. The glucagon derivative of Formula I as described above, wherein X₁₆represents Leu.

30. The glucagon derivative of Formula I as described above, wherein X₁₆represents Thr.

31. The glucagon derivative of Formula I as described above, wherein X₁₆represents Glu.

32. The glucagon derivative of Formula I as described above, wherein X₁₆represents Lys.

33. The glucagon derivative of Formula I as described above, wherein X₁₇represents Arg or Lys.

34. The glucagon derivative of Formula I as described above, wherein X₁₇represents Arg.

35. The glucagon derivative of Formula I as described above, wherein X₁₇represents Lys.

36. The glucagon derivative of Formula I as described above, wherein X₁₈represents Arg, or Ala.

37. The glucagon derivative of Formula I as described above, wherein X₁₈represents Arg.

38. The glucagon derivative of Formula I as described above, wherein X₁₈represents Ala.

39. The glucagon derivative of Formula I as described above, wherein X₂₀represents Gln, Arg, Glu, Aib or Lys.

40. A glucagon derivative of Formula I as described above, wherein X₂₀represents Gln, Arg, Glu, or Lys.

41. The glucagon derivative of Formula I as described above, wherein X₂₀represents Gln.

42. The glucagon derivative of Formula I as described above, wherein X₂₀represents Arg.

43. The glucagon derivative of Formula I as described above, wherein X₂₀represents Glu.

44. The glucagon derivative of Formula I as described above, wherein X₂₀represents Lys.

45. A glucagon derivative of Formula I as described above, wherein X₂₁represents Asp, Glu or Lys.

46. The glucagon derivative of Formula I as described above, wherein X₂₁represents Glu or Lys.

47. The glucagon derivative of Formula I as described above, wherein X₂₁represents Asp.

48. The glucagon derivative of Formula I as described above, wherein X₂₁represents Glu.

49. The glucagon derivative of Formula I as described above, wherein X₂₁represents Lys.

50. A glucagon derivative of Formula I as described above, wherein X₂₄represents Gln, Ala, Arg, Glu, Aib or Lys.

51. A glucagon derivative of Formula I as described above, wherein X₂₄represents Gln, Ala, Arg, or Lys.

52. The glucagon derivative of Formula I as described above, wherein X₂₄represents Gln.

53. The glucagon derivative of Formula I as described above, wherein X₂₄represents Ala.

54. The glucagon derivative of Formula I as described above, wherein X₂₄represents Arg.

55. The glucagon derivative of Formula I as described above, wherein X₂₄represents Lys.

56. A glucagon derivative of Formula I as described above, wherein X₂₇represents Met, Leu or Val.

57. A glucagon derivative of Formula I as described above, wherein X₂₇represents Leu or Val.

58. The glucagon derivative of Formula I as described above, wherein X₂₇represents Met.

59. The glucagon derivative of Formula I as described above, wherein X₂₇represents Leu.

60. The glucagon derivative of Formula I as described above, wherein X₂₇represents Val.

61. A glucagon derivative of Formula I as described above, wherein X₂₈represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys.

62. A glucagon derivative of Formula I as described above, wherein X₂₈represents Asn, Ser, or Lys.

63. The glucagon derivative of Formula I as described above, wherein X₂₈represents Asn.

64. The glucagon derivative of Formula I as described above, wherein X₂₈represents Ser.

65. The glucagon derivative of Formula I as described above, wherein X₂₈represents Lys.

66. A glucagon derivative of Formula I as described above, wherein X₂₉represents Thr, Gly, Ser, Gln, Ala, Glu or Lys.

67. A glucagon derivative of Formula I as described above, wherein X₂₉represents Thr, Gly, or Lys.

68. The glucagon derivative of Formula I as described above, wherein X₂₉represents Gly or Lys.

69. The glucagon derivative of Formula I as described above, wherein X₂₉represents Thr.

70. The glucagon derivative of Formula I as described above, wherein X₂₉represents Gly.

71. The glucagon derivative of Formula I as described above, wherein X₂₉represents Lys.

72. A glucagon derivative of Formula I as described above, wherein X₃₀represents Lys, or wherein X₃₀ is absent.

73. The glucagon derivative of Formula I as described above, wherein X₃₀represents Lys.

74. The glucagon derivative of Formula I as described above, wherein X₃₀is absent.

75. The glucagon derivative of Formula I as described above, wherein

X₂ represents Aib;

X₂₀ represents Arg; and

X₂₁ represents Glu.

76. The glucagon derivative of Formula I as described above, comprisingan amino acid sequence with any one of the following amino acidsubstitutions:

[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28];

[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28];

[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28];

[Aib2,Leu10,Arg20,Leu27,Lys28];

[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Arg20,Leu27,Ser28];

[Aib2,Leu10,Lys16,Lys17,Glu21,Leu27];

[Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29];

[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Lys16,Arg20,Glu21,Leu27];

[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29];

[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29];

[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28];

[Aib2,Leu10,Lys16,Lys17,Ala8,Arg20,Glu21,Ala24,Leu27];

[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27];

[Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27];

[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29];

[Aib2,Leu10,Lys16,Glu21,Leu27];

[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28];

[Acb2,His3,Leu10,Glu15,Leu27,Lys28];

[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28];

[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29];

[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28];

[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28];

[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28];

[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Leu16,Leu27,Lys28];

[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28];

[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28];

[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28];

[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Glu15,Leu27,Lys28];

[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28];

[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29];

[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]; and

[Aib2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28].

77. The glucagon derivative of Formula I as described above, comprisingan amino acid sequence with any one of the following amino acidsubstitutions:

[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28];

[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; and

[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28].

78. The glucagon derivative of Formula I as described above, comprisingan amino acid sequence with any one of the following amino acidsubstitutions:

[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28];

[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; and

[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28].

79. The glucagon derivative of Formula I as described above, comprisingan amino acid sequence with the following amino acid substitutions:

[Aib2,Leu10,Glu15,Lys17,Arg20,Ser21,Leu27,Lys28].

80. The glucagon derivative of Formula I as described above, comprisingan amino acid sequence with the following amino acid substitutions:

[Aib2,Val10,Ala16,Leu27,Lys28].

81. A glucagon derivative of Formula I as described above, the aminoacid sequence comprises a lysine residue at one or more of positions 12,16, 17, 18, 20, 21, 24, 28, 29, and/or 30.

82. The glucagon derivative of Formula I as described above, the aminoacid sequence comprises a lysine residue at one, two or three ofpositions 12, 16, 17, 18, 20, 21, 24, 28, 29, and/or 30.

83. The glucagon derivative of Formula I as described above, the aminoacid sequence comprises a lysine residue at one or two of positions 12,16, 17, 18, 20, 21, 24, 28, 29, and/or 30.

84. The glucagon derivative of Formula I as described above, the aminoacid sequence comprises a lysine residue at two of positions 12, 16, 17,18, 20, 21, 24, 28, 29, and/or 30; in particular in positions 12 and 28.

85. The glucagon derivative of Formula I as described above, the aminoacid sequence comprises a lysine residue at position 12, 16, 17, 18, 20,21, 24, 28, 29, or 30.

86. A glucagon derivative of Formula I as described above, wherein thesubstituent comprising a lipophilic moiety and at least three negativelycharged moieties, is attached at the epsilon position of a lysineresidue in position 16, 21, 24, 28, 29, or 30.

87. The glucagon derivative of Formula I as described above, wherein thesubstituent comprising a lipophilic moiety and at least three negativelycharged moieties, is attached at the epsilon position of a lysineresidue in position 16, 21, 24, 28, 29, or 30.

88. The glucagon derivative of Formula I as described above, wherein thesubstituent comprising a lipophilic moiety and at least three negativelycharged moieties, is attached at the epsilon position of a lysineresidue in position 16, 24, 28, 29 or 30.

89. The glucagon derivative of Formula I as described above, wherein thesubstituent comprising a lipophilic moiety and at least three negativelycharged moieties, is attached at the epsilon position of a lysineresidue in position 24, 28, 29 or 30.

90. The glucagon derivative of Formula I as described above, wherein thesubstituent comprising a lipophilic moiety and at least three negativelycharged moieties, is attached at the epsilon position of a lysineresidue in position 28, 29 and 30.

91. The glucagon derivative of Formula I as described above, wherein thesubstituent comprising a lipophilic moiety and at least three negativelycharged moieties, is attached at the epsilon position of a lysineresidue in position 28.

92. The glucagon derivative of Formula I as described above, wherein thesubstituent comprising a lipophilic moiety and three or more negativelycharged moieties is a substituent of Formula II:

Z¹-Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-

wherein,

Z¹-represents a structure of Formula IIa;

wherein n is 6-20; and the symbol * represents the attachment point tothe nitrogen of the neighbouring linking group; and

Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰- represents a linking group, wherein each ofZ₂ to Z₁₀ individually are represented by any one of the following aminoacid residues: Glu, γGlu, Gly, Ser, Ala, Thr or Ado; or one or more ofresidues Z² to Z¹⁰ are absent; provided, however, that at least two ofresidues Z² to Z¹⁰ are present; and

wherein Z¹-Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-together contains at least threenegative charges; and

wherein said substituent is attached at the epsilon position of a Lysresidue according to Formula I.

93. The glucagon derivative of Formula I as described above, wherein Z¹of Formula II represents a structure according to Formula IIa:

wherein n represents an integer in the range of from 6 to 20;

the symbol * represents the attachment point to the nitrogen of theneighbouring group; and

wherein Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, Z₁₀ individually are representedby the following amino acids: Glu, γGlu, Gly, Ser, Ala, Thr and Ado; orone or more of Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, Z₁₀ may be absent;provided, however, that at least two of residues Z₂ to Z₁₀ are present;

wherein Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀ together contains at least threenegative charges; and

wherein said substituent is attached at the epsilon position of a Lysresidue according to Formula I, above.

94. The glucagon derivative of Formula I as described above, wherein nin Z¹ of Formula IIa represents 14, 16 or 18.

95. The glucagon derivative of Formula I as described above, wherein nin Z¹ of Formula IIa represents 14.

96. The glucagon derivative of Formula I as described above, wherein nin Z¹ of Formula IIa represents 16.

97. The glucagon derivative of Formula I as described above, wherein nin Z¹ of Formula IIa represents 18.

98. The glucagon derivative of Formula I as described above, whereinsaid substituent represents a structure according to any one of thefollowing nine formulas (Chem.A-Chem.I), wherein * indicates the pointof attachment to the nitrogen atom of the epsilon position of a Lysresidue of Formula I:

99. The glucagon derivative of Formula I as described above, whereinsaid substituent represents a structure according to the followingformula (Chem.J), wherein * indicates the point of attachment to thenitrogen atom of the epsilon position of a Lys residue of Formula I:

100. The glucagon derivative of Formula I as described above, whereinsaid substituent represents a structure according to any one of formulasChem.A-Chem.I or Chem.J, as described above, wherein * indicate thepoint of attachment to the nitrogen atom of the epsilon position of aLys residue according to Formula.I.

101. The glucagon derivative of Formula I as described above, whereinsaid substituent represents a structure according to the formula Chem.B,Chem.C, or Chem.H, or according to the formula Chem.J, described above,wherein * indicate the point of attachment to the nitrogen atom of theepsilon position of a Lys residue according to Formula I.

102. A glucagon derivative of the invention selected from the groupconsisting of:

-   N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28]-Glucagon    amide;-   N^(ε21)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28]-Glucagon    amide;-   N^(ε24)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29]-Glucagon    amide;-   N^(α)-([Aib2,Leu10,Arg20,Leu27,Ser28]-Glucagonyl)-N^(ε)[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]Lys    amide;-   N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Glu21,Leu27]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29]-Glucagon    amide;-   N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Leu27]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys6,Glu20,Leu27,Ser28,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29]-Glucagon    amide;-   N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]-acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29]-Glucagon    amide;-   N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Leu27]-Glucagon    amide;-   N^(ε28)[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28]-Glucagon    amide;-   N^(ε16)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,    Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide;-   N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,His3,Leu10,Glu15,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4 S)-4-carboxy-4-[[(4    S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4    S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4 S)-4-carboxy-4-[[(4    S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4    S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Acb2,Leu10,Glu15,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29]-Glucagon    amide;-   N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)    butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]-Glucagon    amide;-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide; and-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon    amide.

103. A glucagon derivative of the invention, wherein said glucagonderivative is selected from the group consisting of:

-   N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Ser21,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Leu16,Leu27,Lys28]-Glucagon    amide;-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Ala16,Leu27,Lys28]-Glucagon    amide; and-   N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Leu16,Leu27,Lys28]-Glucagon    amide.

104. A glucagon derivative of the invention, wherein said glucagonderivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Ala16,Leu27,Lys28]-Glucagonamide.

Further embodiments of the invention relate to:

105. The glucagon derivative of Formula I as described above, whereinsaid glucagon derivative is a GLP-1 and glucagon receptor co-agonist.

106. The glucagon derivative according to any of the previousembodiments, wherein said substituent binds non-covalently to albumin.

107. The glucagon derivative according to any of the previousembodiments, wherein said substituent is negatively charged atphysiological pH.

108. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is a DPPIV protectedcompound.

109. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is DPPIV stabilised.

110. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of theglucagon receptor.

111. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of theglucagon receptor, with an EC₅₀<10 nM.

112. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of theglucagon receptor, with an EC₅₀<1 nM.

113. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of theglucagon receptor, with an EC₅₀<100 pM.

114. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of theglucagon receptor, with an EC₅₀<10 pM.

115. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor.

116. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor, with an EC₅₀<100 pM.

117. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor, with an EC₅₀<50 pM.

118. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor, with an EC₅₀<10 pM.

119. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<100 pM and an EC₅₀<1 nM on the glucagon receptor.

120. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<50 pM and an EC₅₀<1 nM on the glucagon receptor.

121. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<1 nM on the glucagon receptor.

122. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<50 pM and an EC₅₀<100 pM on the glucagon receptor.

123. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<100 pM on the glucagon receptor.

124. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<50 pM on the glucagon receptor.

125. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<1 pM and an EC₅₀<50 pM on the glucagon receptor.

126. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is an agonist of the GLP-1receptor with an EC₅₀<10 pM and an EC₅₀<10 pM on the glucagon receptor.

127. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is a GLP-1/glucagonco-agonist with an EC₅₀ on the GLP-1 receptor<the EC₅₀ on the glucagonreceptor.

128. The glucagon derivative according to any of the previousembodiments, wherein said glucagon derivative is a GLP-1/glucagonco-agonist with an EC₅₀ on the GLP-1 receptor (e.g. in pM) greater thanthe EC₅₀ on the glucagon receptor (e.g. in pM).

Further embodiments of the invention relate to combinations:

129. The glucagon derivative according to any one of the previousembodiments, in combination with a GLP-1 compound or with an insulincompound.

130. The glucagon derivative according to any one of the previousembodiments, in combination with a GLP-1 compound.

131. The glucagon derivative according to any one of the previousembodiments, in combination with an insulin compound.

132. The glucagon derivative according to any one of embodiments123-125, wherein the GLP-1 compound is selected from the groupconsisting of:

-   N-epsilon26-((S)-4-Carboxy-4-hexadecanoylamino-butyryl)    [Arg34]GLP-1-(7-37):

-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg126,Arg34,Lys37]GLP-1-(7-37):

-   N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)    butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37):

and

-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,22,35,Lys37]GLP-1-(7-37):

and their pharmaceutically acceptable salts, amides, alkyls, or esters.

133. The glucagon derivative according to any one of embodiments129-132, wherein the insulin compound is:

N-epsilon-B29-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyryl]desB30 human insulin

Other embodiments of the present relates to pharmaceutical compositions:

134. A pharmaceutical composition comprising a glucagon derivativeaccording to any one of embodiments 1-128 and optionally one or morepharmaceutically acceptable excipients.

135. The pharmaceutical composition according to embodiment 134, furthercomprising one or more additional therapeutically active compounds orsubstances.

136. The pharmaceutical composition according to embodiment 135, whereinsaid additional therapeutically active compound is a GLP-1 compound oran insulin compound.

137. The pharmaceutical composition according to embodiment 136, whereinsaid additional therapeutically active compound is a GLP-1 compound.

138. The pharmaceutical composition according to embodiment 136 or 137,wherein said additional therapeutically active compound is an insulincompound.

139. The pharmaceutical composition according to any one of embodiments136-138, wherein the GLP-1 compound is selected from the groupconsisting of:

-   N-epsilon26-((S)-4-Carboxy-4-hexadecanoylamino-butyryl)[Arg34]GLP-1-(7-37):

-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg126,Arg34,Lys37]GLP-1-(7-37):

-   N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37):

and

-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,22,35,Lys37]GLP-1-(7-37):

and their pharmaceutically acceptable salts, amides, alkyls, or esters.

140. The pharmaceutical composition according to any one of embodiments130-133, wherein the insulin compound is:

N-epsilon-B29-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyryl]desB30 human insulin

141. The pharmaceutical composition according to any one of embodiments128-134, in unit dosage form comprising from about 0.01 mg to about 1000mg, such as from about 0.1 mg to about 500 mg, from about 0.5 mg toabout 5 mg, e.g. from about 0.5 mg to about 200 mg, of a glucagonderivative according to any of embodiments 1-128.

142. The pharmaceutical composition according to any one of embodiments126-138, which is suited for parenteral administration.

143. The glucagon derivative according to any one of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in therapy.

Further embodiments of the invention relate to the following:

144. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treatment or prevention of hyperglycemia,type 2 diabetes, impaired glucose tolerance, type 1 diabetes andobesity.

145. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in delaying or preventing disease progressionin type 2 diabetes.

146. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use treating obesity or preventing overweight.

147. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in for decreasing food intake.

148. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in increasing energy expenditure.

149. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in reducing body weight.

150. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in delaying the progression from impairedglucose tolerance (IGT) to type 2 diabetes.

151. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in delaying the progression from type 2diabetes to insulin-requiring diabetes.

152. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in regulating appetite.

153. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in inducing satiety.

154. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in preventing weight regain after successfulweight loss.

155. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating a disease or state related tooverweight or obesity.

156. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating bulimia.

157. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating binge-eating.

158. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating atherosclerosis.

159. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating hypertension.

160. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating type 2 diabetes.

161. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating impaired glucose tolerance.

162. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating dyslipidemia.

163. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating coronary heart disease.

164. The glucagon derivative according to any of embodiments 1-128,optionally in combination with one or more additional therapeuticallyactive compounds, for use in treating hepatic steatosis.

Further embodiments of the invention relate to the following methods:

165. A method for treating or preventing hypoglycemia, type 2 diabetes,impaired glucose tolerance, type 1 diabetes and obesity, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

166. A method for delaying or preventing disease progression in type 2diabetes, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative according to any ofembodiments 1-128, optionally in combination with one or more additionaltherapeutically active compounds.

167. A method for treating obesity or preventing overweight, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

168. A method for decreasing food intake, comprising administering to apatient in need thereof, an effective amount of a glucagon derivativeaccording to any of embodiments 1-128, optionally in combination withone or more additional therapeutically active compounds.

169. A method for use in increasing energy expenditure, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

170. A method for use in reducing body weight, comprising administeringto a patient in need thereof, an effective amount of a glucagonderivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

171. A method for use in delaying the progression from impaired glucosetolerance (IGT) to type 2 diabetes, comprising administering to apatient in need thereof, an effective amount of a glucagon derivativeaccording to any of embodiments 1-128, optionally in combination withone or more additional therapeutically active compounds.

172. A method for use in delaying the progression from type 2 diabetesto insulin-requiring diabetes, comprising administering to a patient inneed thereof, an effective amount of a glucagon derivative according toany of embodiments 1-128, optionally in combination with one or moreadditional therapeutically active compounds.

173. A method for use in regulating appetite, comprising administeringto a patient in need thereof, an effective amount of a glucagonderivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

174. A method for use in inducing satiety, comprising administering to apatient in need thereof, an effective amount of a glucagon derivativeaccording to any of embodiments 1-128, optionally in combination withone or more additional therapeutically active compounds.

175. A method for use in preventing weight regain after successfulweight loss, comprising administering to a patient in need thereof, aneffective amount of a glucagon derivative according to any ofembodiments 1-128, optionally in combination with one or more additionaltherapeutically active compounds.

176. A method for use in treating a disease or state related tooverweight or obesity, comprising administering to a patient in needthereof, an effective amount of a glucagon derivative according to anyof embodiments 1-128, optionally in combination with one or moreadditional therapeutically active compounds.

177. A method for use in treating bulimia, comprising administering to apatient in need thereof, an effective amount of a glucagon derivativeaccording to any of embodiments 1-128, optionally in combination withone or more additional therapeutically active compounds.

178. A method for use in treating binge-eating, comprising administeringto a patient in need thereof, an effective amount of a glucagonderivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

179. A method for use in treating atherosclerosis, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

180. A method for use in treating hypertension, comprising administeringto a patient in need thereof, an effective amount of a glucagonderivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

181. A method for use in treating type 2 diabetes, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

182. A method for use in treating impaired glucose tolerance, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

183. A method for use in treating dyslipidemia, comprising administeringto a patient in need thereof, an effective amount of a glucagonderivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

184. A method for use in treating coronary heart disease, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

185. A method for use in treating hepatic steatosis, comprisingadministering to a patient in need thereof, an effective amount of aglucagon derivative according to any of embodiments 1-128, optionally incombination with one or more additional therapeutically activecompounds.

Further embodiments of the invention relate to the following uses:

186. Use of a glucagon derivative according to any one of theembodiments 1-128, for the preparation of a medicament.

187. Use of a glucagon derivative according to any one of embodiments1-128, for the preparation of a medicament for the treatment orprevention of hyperglycemia, type 2 diabetes, impaired glucosetolerance, type 1 diabetes and obesity.

188. Use of a glucagon derivative according to any one of theembodiments 1-128, for the preparation of a medicament for delaying orpreventing disease progression in type 2 diabetes, treating obesity orpreventing overweight, for decreasing food intake, increase energyexpenditure, reducing body weight, delaying the progression fromimpaired glucose tolerance (IGT) to type 2 diabetes; delaying theprogression from type 2 diabetes to insulin-requiring diabetes;regulating appetite; inducing satiety; preventing weight regain aftersuccessful weight loss; treating a disease or state related tooverweight or obesity; treating bulimia; treating binge-eating; treatingatherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia,coronary heart disease, hepatic steatosis, treatment of beta-blockerpoisoning, use for inhibition of the motility of the gastrointestinaltract, useful in connection with investigations of the gastrointestinaltract using techniques such as x-ray, CT- and NMR-scanning.

Further embodiments of the invention relate to the following:

189. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has more than 70% recovery in the ThTfibrillation assay.

190. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has more than 90% recovery in the ThTfibrillation assay.

191. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has about 100% recovery in the ThTfibrillation assay.

192. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has more than 7 hours lag time in theThT fibrillation assay.

193. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has more than 20 hours lag time in theThT fibrillation assay.

194. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has 45 hours lag time or more in theThT fibrillation assay.

195. A glucagon derivative according to any of the previous embodiments,wherein said ThT fibrillation assay is as described in Example 76herein.

196. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 14% degradation in thechemical stability assay.

197. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 13% degradation in thechemical stability assay.

198. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 12% degradation in thechemical stability assay.

199. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 10% degradation in thechemical stability assay.

200. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 9% degradation in thechemical stability assay.

201. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 7% degradation in thechemical stability assay.

202. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 5% degradation in thechemical stability assay.

203. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 3% degradation in thechemical stability assay.

204. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative has less than 2% degradation in thechemical stability assay.

205. A glucagon derivative according to any of the previous embodiments,wherein said chemical stability assay is as described in Example 79herein.

206. A glucagon derivative according to any of the previous embodiments,wherein said glucagon derivative is as selective for glucagon receptoras for GLP-1 receptor.

207. A glucagon derivative as defined in any of the previousembodiments, optionally in combination with one or more additionaltherapeutically active compounds, for use in medicine.

208. The glucagon derivative according to embodiment 207, optionally incombination with one or more additional therapeutically activecompounds, for i) treating obesity, preventing overweight, and/orreducing body weight, and/or ii) treatment or prevention ofhyperglycemia, type 2 diabetes, impaired glucose tolerance, and/or type1 diabetes.

Further Embodiments of the Invention

The invention can further be described by the following furthernon-limiting embodiments:

-   1. A glucagon derivative comprising (formula I) (SEQ ID NO:2 and SEQ    ID NO:3):    His-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₁₇-X₁₈-Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀    (formula I), wherein

X₂ is Aib, Acb or Acpr, X₃ is Gln or His, X₁₀ is Leu, Ile or Val, X₁₂ isLys or Arg, X₁₅ is Asp or Glu X₁₆ is Ser, Ala, Leu, Thr, Glu, Aib, Ile,Val or Lys, X₁₇ is Arg or Lys, X₁₈ is Arg, Ala or Lys, X₂₀ is Gln, Arg,Glu, Aib or Lys, X₂₁ is Asp, Glu or Lys, X₂₄ is Gln, Ala, Arg, Glu, Aibor Lys, X₂₇ is Met, Leu or Val, X₂₈ is Asn, Ser, Thr, Gln, Ala, Gly, Gluor Lys, X₂₉ is Thr, Gly, Ser, Gln, Ala, Glu or Lys,

X₃₀ is absent or is Lys,and a substituent comprising a lipophilic moiety and three or morenegatively charged moieties, wherein one of said negatively chargedmoieties is distal of a lipophilic moiety, and wherein said substituentis attached at the epsilon position of a Lys in one of the followingamino acid positions of said glucagon derivative: 16, 17, 18, 20, 21,24, 28, 29, and/or 30, wherein said glucagon derivative is a C-terminalamide or a pharmaceutically acceptable salt or prodrug thereof.

-   2. The glucagon derivative according to embodiment 1, wherein said    glucagon derivative is a GLP-1 and glucagon receptor co-agonist.-   3. The glucagon derivative according to embodiment 2, wherein X₂ is    Aib.-   4. The glucagon derivative according to embodiment 2, wherein X₂ is    Acb.-   5. The glucagon derivative according to embodiment 2, wherein X₂ is    Acpr.-   6. The glucagon derivative according to any one of the previous    embodiments, wherein X₃ is Gln.-   7. The glucagon derivative according to any one of the previous    embodiments, wherein X₃ is His.-   8. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₀ is Leu.-   9. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₀ is Ile.-   10. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₀ is Val.-   11. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₂ is Lys.-   12. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₂ is Arg.-   13. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₅ is Asp.-   14. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₅ is Glu.-   15. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₆ is Ser, Ala, Leu, Thr, Glu or Lys.-   16. The glucagon derivative according to embodiment 15, wherein X₁₆    is Ser.-   17. The glucagon derivative according to embodiment 15, wherein X₁₆    is Ala, Leu, Thr, Glu or Lys.-   18. The glucagon derivative according to embodiment 15, wherein X₁₆    is Ala.-   19. The glucagon derivative according to embodiment 15, wherein X₁₆    is Leu.-   20. The glucagon derivative according to embodiment 15, wherein X₁₆    is Thr.-   21. The glucagon derivative according to embodiment 15, wherein X₁₆    is Glu.-   22. The glucagon derivative according to embodiment 15, wherein X₁₆    is Lys.-   23. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₇ is Arg.-   24. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₇ is Lys.-   25. The glucagon derivative according to any one of the previous    embodiments, wherein X₁₈ is Arg or Ala.-   26. The glucagon derivative according to embodiment 25, wherein X₁₈    is Arg.-   27. The glucagon derivative according to embodiment 25, wherein X₁₈    is Ala.-   28. The glucagon derivative according to any one of the previous    embodiments, wherein X₂₀ is Gln, Arg, Glu or Lys.-   29. The glucagon derivative according to embodiment 28, wherein X₂₀    is Gln.-   30. The glucagon derivative according to embodiment 28, wherein X₂₀    is Arg.-   31. The glucagon derivative according to embodiment 28, wherein X₂₀    is Glu.-   32. The glucagon derivative according to embodiment 28, wherein X₂₀    is Lys.-   33. The glucagon derivative according to any one of the previous    embodiments, wherein X₂₁ is Asp.-   34. The glucagon derivative according to any one of the previous    embodiments, wherein X₂₁ is Glu or Lys.-   35. The glucagon derivative according to embodiment 34, wherein X₂₁    is Glu.-   36. The glucagon derivative according to embodiment 34, wherein X₂₁    is Lys.-   37. The glucagon derivative according to any one of the previous    embodiments, wherein X₂₄ is Gln, Ala, Arg or Lys.-   38. The glucagon derivative according to embodiment 37, wherein X₂₄    is Gln.-   39. The glucagon derivative according to embodiment 37, wherein X₂₄    is Ala.-   40. The glucagon derivative according to embodiment 37, wherein X₂₄    is Arg.-   41. The glucagon derivative according to embodiment 37, wherein X₂₄    is Lys.-   42. The glucagon derivative according to any one of the previous    embodiments, wherein X₂₇ is Met, Leu or Val.-   43. The glucagon derivative according to embodiment 42, wherein X₂₇    is Met.-   44. The glucagon derivative according to embodiment 42, wherein X₂₇    is Leu.-   45. The glucagon derivative according to embodiment 42, wherein X₂₇    is Val.-   46. The glucagon derivative according to any one of the previous    embodiments, wherein X₂₈ is Asn, Ser or Lys.-   47. The glucagon derivative according to embodiment 46, wherein X₂₈    is Asn.-   48. The glucagon derivative according to embodiment 46, wherein X₂₈    is Ser.-   49. The glucagon derivative according to embodiment 46, wherein X₂₈    is Lys.-   50. The glucagon derivative according to any one of the previous    embodiments, wherein X₂₉ is Thr, Gly or Lys.-   51. The glucagon derivative according to embodiment 50, wherein X₂₉    is Gly or Lys.-   52. The glucagon derivative according to embodiment 50, wherein X₂₉    is Thr.-   53. The glucagon derivative according to embodiment 50, wherein X₂₉    is Gly.-   54. The glucagon derivative according to embodiment 50, wherein X₂₉    is Lys.-   55. The glucagon derivative according to any one of the previous    embodiments, wherein X₃₀ is absent.-   56. The glucagon derivative according to any one of embodiments    1-54, wherein X₃₀ is Lys.-   57. The glucagon derivative according to any one of the previous    embodiments, wherein X₂ represents Aib, X₂₀ represents Arg and X₂₁    represents Glu.-   58. The glucagon derivative according to any of the previous    embodiments, wherein the amino acid substitutions are:

(i) 2Acb, 10L, 12R, 16L, 20R, 27L, 28K;

(ii) 2Acb, 10L, 15E, 16E, 20R, 27L 28K;

(iii) 2Acb, 10L, 15E, 17K, 20R, 21E, 27L, 28K;

(iv) 2Acb, 10L, 15E, 20R, 21E, 27L, 28K;

(v) 2Acb, 10L, 16L, 17K, 20R, 21E, 27L, 28K;

(vi) 2Acb, 10L, 16L, 20R, 21E, 27L, 28K;

(vii) 2Acb, 10L, 16L, 20R, 27L, 28K;

(viii) 2Acb, 3H, 10L, 15E, 27L, 28K;

(ix) 2Acpr, 10L, 15E, 20R, 21E, 27L, 28K;

(x) 2Aib, 10L, 15E, 17K, 18A, 20R, 21E, 27L, 28K;

(xi) 2Aib, 10L, 15E, 17K, 20R, 21E, 27L, 28K;

(xii) 2Aib, 10L, 15E, 20R, 21E, 27L, 28K;

(xiii) 2Aib, 10L, 16A, 20R, 21E, 27L, 28K;

(xiv) 2Aib, 10L, 16E, 20K, 27L, 28S, 29K;

(xv) 2Aib, 10L, 16K, 17K, 18A, 20R, 21E, 24A, 27L;

(xvi) 2Aib, 10L, 16K, 17K, 21E, 27L;

(xvii) 2Aib, 10L, 16K, 18A, 20R, 21E, 24A, 27L;

(xviii) 2Aib, 10L, 16K, 20E, 27L, 28S, 29K;

(xix) 2Aib, 10L, 16K, 20R, 21E, 24A, 27L;

(xx) 2Aib, 10L, 16K, 20R, 21E, 27L;

(xxi) 2Aib, 10L, 16K, 20R, 27L, 28S;

(xxii) 2Aib, 10L, 16K, 21E, 27L;

(xxiii) 2Aib, 10L, 16K, 21E, 27V, 28K, 29G;

(xxiv) 2Aib, 10L, 16L, 17K, 20R, 21E, 27L, 28K;

(xxv) 2Aib, 10L, 16L, 20R, 21E, 27L, 29K;

(xxvi) 2Aib, 10L, 16T, 20K, 27L, 28S, 29K;

(xxvii) 2Aib, 10L, 16T, 24R, 27L, 28S, 29K;

(xxviii) 2Aib, 10L, 17K, 18A, 21E, 27L, 29K;

(xxix) 2Aib, 10L, 18A, 20R, 21E, 27L, 29K;

(xxx) 2Aib, 10L, 20R, 21E, 231, 27L, 29K;

(xxxi) 2Aib, 10L, 20R, 21E, 27L, 29K;

(xxxii) 2Aib, 10 L, 20R, 21K, 27L, 28S;

(xxxiii) 2Aib, 10L, 20R, 24K, 27L, 28S;

(xxxiv) 2Aib, 10L, 20R, 27L, 28K;

(xxxv) 2Aib, 10L, 20R, 27L, 28S, 29K;

(xxxvi) 2Aib, 10L, 20R, 27L, 28S, 30K;

(xxxvii) 2Aib, 3H, 10L, 15E, 16K, 20R, 21E, 24A, 27L, 28S;

(xxxviii) 2Aib, 3H, 10L, 15E, 17K, 20R, 21E, 27L, 28K;

(xxxix) 2Aib, 3H, 10L, 15E, 20R, 21E, 24A, 27L, 28K;

(xl) 2Aib, 3H, 10L, 15E, 20R, 21E, 27L, 28K or

(xli) 2Aib, 3H, 10L, 15E, 20R, 27L, 28K.

-   59. The glucagon derivative according to any one of the previous    embodiments, wherein said substituent has formula II:

Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-  [II]

wherein,Z₁ represents a structure according to the formula IIa;

wherein n in formula IIa is 6-20,the symbol * in formula Z₁ represents the attachment point to thenitrogen of the neighbouring group and wherein Z₂, Z₃, Z₄, Z₅, Z₆, Z₇,Z₈, Z₉, Z₁₀ individually are represented by the following amino acids;Glu, γGlu, Gly, Ser, Ala, Thr, Ado or are absent,wherein at least two of residues Z₂ to Z₁₀ are present,wherein said substituent is attached at the epsilon position of a Lys offormula I and wherein Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀ together containsat least three negative charges.

-   60. The glucagon derivative according to embodiment 59, wherein n in    Z1 of formula II is 14, 16 or 18.-   61. The glucagon derivative according to embodiment 59, wherein n in    Z1 of formula II is 14.-   62. The glucagon derivative according to embodiment 59, wherein n in    Z1 of formula II is 16.-   63. The glucagon derivative according to embodiment 59, wherein n in    Z1 of formula II is 18.-   64. The glucagon derivative according to any one of the previous    embodiments, wherein said substituent represents a structure    according to one of the formulas and wherein * indicates the point    of attachment to the nitrogen at the epsilon position of a Lys of    formula I:

-   65. The glucagon derivative according to any of the previous    embodiments, wherein said substituent binds non-covalently to    albumin.-   66. The glucagon derivative according to any of the previous    embodiments, wherein said substituent is negatively charged at    physiological pH.-   67. The glucagon derivative according to any one of the previous    embodiments, selected from the group consisting of: Chem.1; Chem.2;    Chem.3; Chem.4; Chem.5; Chem.6; Chem.7; Chem.8; Chem.9; Chem.10;    Chem.11; Chem.12; Chem.13; Chem.14; Chem.15; Chem.16; Chem.17;    Chem.18; Chem.19; Chem.20; Chem.21; Chem.22; Chem.23; Chem.24;    Chem.25; Chem.26; Chem.27; Chem.28; Chem.29; Chem.30; Chem.31;    Chem.32; Chem.33; Chem.34; Chem.35; Chem.36; Chem.37; Chem.38;    Chem.39; Chem.40; Chem.41; Chem.42; Chem.43; Chem.44; Chem.45;    Chem.46; Chem.47; Chem.48; Chem.49 and Chem.50.    Further embodiments of the invention relate to:-   68. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is a DPPIV protected    compound.-   69. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is DPPIV stabilised.-   70. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    glucagon receptor.-   71. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    glucagon receptor, with an EC50<10 nM.-   72. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    glucagon receptor, with an EC50<1 nM.-   73. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    glucagon receptor, with an EC50<100 pM.-   74. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    glucagon receptor, with an EC50<10 pM.-   75. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor.-   76. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor, with an EC50<100 pM.-   77. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor, with an EC50<50 pM.-   78. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor, with an EC50<10 pM.-   79. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor with an EC50<100 pM and an EC50<1 nM on the glucagon    receptor.-   80. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor with an EC50<50 pM and an EC50<1 nM on the glucagon    receptor.-   81. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor with an EC50<10 pM and an EC50<1 nM on the glucagon    receptor.-   82. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor with an EC50<50 pM and an EC50<100 pM on the glucagon    receptor.-   83. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor with an EC50<10 pM and an EC50<100 pM on the glucagon    receptor.-   84. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor with an EC50<10 pM and an EC50<50 pM on the glucagon    receptor.-   85. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is an agonist of the    GLP-1 receptor with an EC50<10 pM and an EC50<10 pM on the glucagon    receptor.-   86. The glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is a GLP-1/glucagon    co-agonist with an EC50 on the GLP-1 receptor<the EC50 on the    glucagon receptor.    Further embodiments of the invention relate to combinations:-   87. The glucagon derivative according to any one of the previous    embodiments, in combination with a GLP-1 compound or with an insulin    compound.-   88. The glucagon derivative according to any one of the previous    embodiments, in combination with a GLP-1 compound.-   89. The glucagon derivative according to any one of the previous    embodiments, in combination with an insulin compound.-   90. The glucagon derivative according to embodiment 88, wherein the    GLP-1 compound is selected from the group consisting of:-   N^(ε26)-((S)-4-Carboxy-4-hexadecanoylamino-butyryl)[Arg34]GLP-1-(7-37):

-   N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)    butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg126,Arg34,Lys37]GLP-1-(7-37):

-   N^(ε26)-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37):

-   N^(ε37)-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,22,35,Lys37]GLP-1-(7-37):

and their pharmaceutically acceptable salts, amides, alkyls, or esters.

-   91. The glucagon derivative according to embodiment 89, wherein the    insulin compound is:    N-epsilon-B29-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyryl]desB30    human insulin

Other embodiments of the present relates to pharmaceutical compositions:

-   92. A pharmaceutical composition comprising a glucagon derivative    according to any one of embodiments 1-91.-   93. The pharmaceutical composition according to embodiment 92,    further comprising one or more additional therapeutically active    compounds or substances.-   94. The pharmaceutical composition according to embodiment 93,    wherein said additional therapeutically active compound is a GLP-1    compound or an insulin compound.-   95. The pharmaceutical composition according to embodiment 94,    wherein said additional therapeutically active compound is a GLP-1    compound.-   96. The pharmaceutical composition according to embodiment 94,    wherein said additional therapeutically active compound is an    insulin compound.-   97. The pharmaceutical composition according to embodiment 95,    wherein the GLP-1 compound is selected from the group consisting of:-   N-epsilon26-((S)-4-Carboxy-4-hexadecanoylamino-butyryl)[Arg34]GLP-1-(7-37):

-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)    butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg126,Arg34,Lys37]GLP-1-(7-37):

-   N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37):

-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,22,35,Lys37]GLP-1-(7-37):

and their pharmaceutically acceptable salts, amides, alkyls, or esters.

-   98. The pharmaceutical composition according to embodiment 96,    wherein the insulin compound is:    N-epsilon-B29-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyryl]desB30    human insulin

-   99. The pharmaceutical composition according to any one of    embodiments 92-98, in unit dosage form comprising from about 0.01 mg    to about 1000 mg, such as from about 0.1 mg to about 500 mg, from    about 0.5 mg to about 5 mg, e.g. from about 0.5 mg to about 200 mg,    of a glucagon derivative according to any of embodiments 1-91.-   100. The pharmaceutical composition according to any one of    embodiments 92-99, which is suited for parenteral administration.-   101. The glucagon derivative according to any one of embodiments    1-91, for use in therapy.    Further embodiments of the invention relate to the following:-   102. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treatment or prevention    of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type    1 diabetes and obesity.-   103. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in delaying or preventing    disease progression in type 2 diabetes.-   104. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use treating obesity or    preventing overweight.-   105. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in for decreasing food    intake.-   106. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in increasing energy    expenditure.-   107. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in reducing body weight.-   108. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in delaying the    progression from impaired glucose tolerance (IGT) to type 2    diabetes.-   109. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in delaying the    progression from type 2 diabetes to insulin-requiring diabetes.-   110. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use regulating appetite.-   111. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use inducing satiety.-   112. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in preventing weight    regain after successful weight loss.-   113. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating a disease or    state related to overweight or obesity.-   114. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating bulimia.-   115. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating binge-eating.-   116. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating    atherosclerosis.-   117. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating hypertension.-   118. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating type 2    diabetes.-   119. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating impaired    glucose tolerance.-   120. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating dyslipidemia.-   121. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating coronary heart    disease.-   122. The glucagon derivative according to any of embodiments 1-91,    optionally in combination with one or more additional    therapeutically active compounds, for use in treating hepatic    steatosis.    Further embodiments of the invention relate to the following    methods:-   123. A method for treating or preventing hypoglycemia, type 2    diabetes, impaired glucose tolerance, type 1 diabetes and obesity,    comprising administering to a patient in need thereof, an effective    amount of a glucagon derivative according to any of embodiments    1-91, optionally in combination with one or more additional    therapeutically active compounds.-   124. A method for delaying or preventing disease progression in type    2 diabetes, comprising administering to a patient in need thereof,    an effective amount of a glucagon derivative according to any of    embodiments 1-91, optionally in combination with one or more    additional therapeutically active compounds.-   125. A method for treating obesity or preventing overweight,    comprising administering to a patient in need thereof, an effective    amount of a glucagon derivative according to any of embodiments    1-91, optionally in combination with one or more additional    therapeutically active compounds.-   126. A method for decreasing food intake, comprising administering    to a patient in need thereof, an effective amount of a glucagon    derivative according to any of embodiments 1-91, optionally in    combination with one or more additional therapeutically active    compounds.-   127. A method for use in increasing energy expenditure, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   128. A method for use in reducing body weight, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   129. A method for use in delaying the progression from impaired    glucose tolerance (IGT) to type 2 diabetes, comprising administering    to a patient in need thereof, an effective amount of a glucagon    derivative according to any of embodiments 1-91, optionally in    combination with one or more additional therapeutically active    compounds.-   130. A method for use in delaying the progression from type 2    diabetes to insulin-requiring diabetes, comprising administering to    a patient in need thereof, an effective amount of a glucagon    derivative according to any of embodiments 1-91, optionally in    combination with one or more additional therapeutically active    compounds.-   131. A method for use in regulating appetite, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   132. A method for use in inducing satiety, comprising administering    to a patient in need thereof, an effective amount of a glucagon    derivative according to any of embodiments 1-91, optionally in    combination with one or more additional therapeutically active    compounds.-   133. A method for use in preventing weight regain after successful    weight loss, comprising administering to a patient in need thereof,    an effective amount of a glucagon derivative according to any of    embodiments 1-91, optionally in combination with one or more    additional therapeutically active compounds.-   134. A method for use in treating a disease or state related to    overweight or obesity, comprising administering to a patient in need    thereof, an effective amount of a glucagon derivative according to    any of embodiments 1-91, optionally in combination with one or more    additional therapeutically active compounds.-   135. A method for use in treating bulimia, comprising administering    to a patient in need thereof, an effective amount of a glucagon    derivative according to any of embodiments 1-91, optionally in    combination with one or more additional therapeutically active    compounds.-   136. A method for use in treating binge-eating, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   137. A method for use in treating atherosclerosis, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   138. A method for use in treating hypertension, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   139. A method for use in treating type 2 diabetes, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   140. A method for use in treating impaired glucose tolerance,    comprising administering to a patient in need thereof, an effective    amount of a glucagon derivative according to any of embodiments    1-91, optionally in combination with one or more additional    therapeutically active compounds.-   141. A method for use in treating dyslipidemia, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   142. A method for use in treating coronary heart disease, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.-   143. A method for use in treating hepatic steatosis, comprising    administering to a patient in need thereof, an effective amount of a    glucagon derivative according to any of embodiments 1-91, optionally    in combination with one or more additional therapeutically active    compounds.    Further embodiments of the invention relate to the following uses:-   144. Use of a glucagon derivative according to any one of the    embodiments 1-91, for the preparation of a medicament.-   145. Use of a glucagon derivative according to any one of    embodiments 1-91, for the preparation of a medicament for the    treatment or prevention of hyperglycemia, type 2 diabetes, impaired    glucose tolerance, type 1 diabetes and obesity.-   146. Use of a glucagon derivative according to any one of the    embodiments 1-91, for the preparation of a medicament for delaying    or preventing disease progression in type 2 diabetes, treating    obesity or preventing overweight, for decreasing food intake,    increase energy expenditure, reducing body weight, delaying the    progression from impaired glucose tolerance (IGT) to type 2    diabetes; delaying the progression from type 2 diabetes to    insulin-requiring diabetes; regulating appetite; inducing satiety;    preventing weight regain after successful weight loss; treating a    disease or state related to overweight or obesity; treating bulimia;    treating binge-eating; treating atherosclerosis, hypertension, type    2 diabetes, IGT, dyslipidemia, coronary heart disease, hepatic    steatosis, treatment of beta-blocker poisoning, use for inhibition    of the motility of the gastrointestinal tract, useful in connection    with investigations of the gastrointestinal tract using techniques    such as x-ray, CT- and NMR-scanning.    Further embodiments of the invention relate to the following:-   147. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has more than 70%    recovery in the ThT fibrillation assay.-   148. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has more than 90%    recovery in the ThT fibrillation assay.-   149. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has about 100%    recovery in the ThT fibrillation assay.-   150. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has more than 7 hours    lag time in the ThT fibrillation assay.-   151. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has more than 20 hours    lag time in the ThT fibrillation assay.-   152. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has 45 hours lag time    or more in the ThT fibrillation assay.-   153. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 14%    degradation in the chemical stability assay.-   154. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 13%    degradation in the chemical stability assay.-   155. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 12%    degradation in the chemical stability assay.-   156. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 10%    degradation in the chemical stability assay.-   157. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 9%    degradation in the chemical stability assay.-   158. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 7%    degradation in the chemical stability assay.-   159. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 5%    degradation in the chemical stability assay.-   160. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 3%    degradation in the chemical stability assay.-   161. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative has less than 2%    degradation in the chemical stability assay.-   162. A glucagon derivative according to any of the previous    embodiments, wherein said glucagon derivative is as selective for    glucagon receptor as for GLP-1 receptor.

EXAMPLES

The invention is further illustrated with reference to the followingexamples, which are not intended to be in any way limiting to the scopeof the invention as claimed.

LIST OF ABBREVIATIONS

BOC: tert-Butyl oxycarbonyl

DCM: Dichloromethane

DIC: Diisopropylcarbodiimide

Fmoc: 9-fluorenylmethyloxycarbonyl

HOAt: 1-hydroxy-7-azabenzotriazole

HPLC: High Performance Liquid Chromatography

LCMS: Liquid Chromatography Mass Spectroscopy

MeCN: Acetonitrile

Mtt: 4-Methyltrityl

NMP: N-methyl pyrrolidone

Oxyma Pure: Cyano-hydroxyimino-acetic acid ethyl ester

RP: Reverse Phase

RP-HPLC: Reverse Phase High Performance Liquid Chromatography

RT: Room Temperature

Rt: Retention time

SPPS: Solid Phase Peptide Synthesis

TFA: Trifluoroacetic acid

TIPS: Triisopropylsilane

UPLC: Ultra Performance Liquid Chromatography

General Methods

This section relates to methods for synthesising resin bound peptide(SPPS methods, including methods for de-protection of amino acids,methods for cleaving the peptide from the resin, and for itspurification), as well as methods for detecting and characterising theresulting peptide (LCMS and UPLC methods).

The compounds in Examples 1-73 herein were prepared, purified andanalysed according to the procedures described below.

SPPS General Methods

The Fmoc-protected amino acid derivatives used were the standardrecommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-lle-OH,Fmoc-Leu-OH, Fmoc-Lys(BOC)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(BOC)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Val-OH and Fmoc-Lys(Mtt)-OH supplied from e.g. Anaspec, Bachem,Iris Biotech, or NovabioChem.

The N-terminal amino acid is Boc protected at the alpha amino group(e.g. Boc-His(Trt)-OH for peptides with His at the N-terminus).

The introduction of the substituent on the epsilon-nitrogen of a lysinewas achieved using a Lysine protected with Mtt (Fmoc-Lys(Mtt)-OH).Suitably protected building blocks such asFmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-Glu-OtBu as well as theprotected standard amino acids described above were used for theintroduction of the substituent. Introduction of the fatty acid moietywas achieved using octadecanedioic acid mono-tert-butyl-ester.

SPPS was performed on a Prelude Solid Phase Peptide Synthesizer fromProtein Technologies (Tucson, Ariz. 85714 U.S.A.) at 100 or 250-μmolscale using six fold excess of Fmoc-amino acids (300 mM in NMP with 300mM HOAt or Oxyma Pure®) relative to resin loading. H-RinkAmide-ChemMatrix resin (loading e.g. 0.52 nmol/g) or Rink Amide AMpolystyrene resin (Novabiochem, loading e.g. 0.62 mmol/g) was used asthe solid support. Fmoc-deprotection was performed using 20% piperidinein NMP. Coupling was performed using 3:3:3:4 amino acid/(HOAt or OxymaPure®)/DIC/collidine in NMP. NMP and DCM top washes (7 ml, 0.5 min, 2×2each) were performed between deprotection and coupling steps. Couplingtimes were generally 60 minutes. Some amino acids including, but notlimited to Fmoc-Arg(Pbf)-OH, Fmoc-Aib-OH or Boc-His(Trt)-OH as well asthe building blocks leading to the substituent were “double coupled”,meaning that after the first coupling (e.g. 60 min), the resin isdrained and more reagents are added (amino acid, (HOAt or Oxyma Pure®),DIC, and collidine), and the mixture allowed to react again (e.g. 60min). The Mtt group was removed by washing the resin with HFIP/DCM(75:25) (2×2 min), washed with DCM and suspending the resin in HFIP/DCM(75:25)(2×20 min) and subsequently washed in sequence withPiperidine/NMP (20:80), DCM(1×), NMP(1×), DCM(1×), NMP(1×).

Cleavage from the Resin

After synthesis the resin was washed with DCM, and the peptide wascleaved from the resin by a 2-3 hour treatment with TFA/TIS/water(95/2.5/2.5) followed by precipitation with diethylether. Theprecipitate was washed with diethylether.

Purification and Quantification

The crude peptide was dissolved in a suitable mixture of water and MeCNsuch as water/MeCN (4:1) and purified by reversed-phase preparative HPLC(Waters Deltaprep 4000 or Gilson) on a column containing C18-silica gel.Elution was performed with an increasing gradient of MeCN in watercontaining 0.1% TFA. Relevant fractions are checked by analytical HPLCor UPLC. Fractions containing the pure target peptide were pooled andconcentrated under reduced pressure. The resulting solution was analyzed(HPLC, LCMS) and the product is quantified using a chemiluminescentnitrogen specific HPLC detector (Antek 8060 HPLC-CLND) or by measuringUV-absorption at 280 nm. The product is dispensed into glass vials. Thevials are capped with Millipore glassfibre prefilters. Freeze-dryingaffords the peptide trifluoroacetate as a white solid.

Methods for Detection and Characterization LCMS Methods Method: LCMS 4

LCMS_4 was performed on a setup consisting of Waters Acquity UPLC systemand LCT Premier XE mass spectrometer from Micromass.

Eluents:

A: 0.1% Formic acid in water

B: 0.1% Formic acid in acetonitrile

The analysis was performed at RT by injecting an appropriate volume ofthe sample (preferably 2-10 μl) onto the column which was eluted with agradient of A and B. The UPLC conditions, detector settings and massspectrometer settings were: Column: Waters Acquity UPLC BEH, C-18, 1.7μm, 2.1 mm×50 mm. Gradient: Linear 5%-95% acetonitrile during 4.0 min(alternatively 8.0 min) at 0.4 ml/min. Detection: 214 nm(derivative/analogue output from TUV (Tunable UV detector)) MSionisation mode: API-ES

Scan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu.

Method: LCMS01v01

System LC-system: Waters Acquity UPLC Column: Waters Acquity UPLC BEH,C-18, 1.7 μm, 2.1 mm × 50 mm Detector: Waters (Micromass) LCT Premier XEDetector Ionisation method: ES setup Scanning range: 500-2000 amuOperating mode: W mode positive/negative: positive mode Cone Voltage: 50V Scantime 1 Interscandelay: 0.0 Conditions Linear gradient: 5% to 95% BGradient run-time: 4.0 minutes Total run-time: 7.0 minutes Flow rate:0.4 ml/min Column temperature: 40° C. Eluents Solvent A: 99.90%MQ-water, 0.1% formic acid Solvent B: 99.90% acetonitrile, 0.1% formicacid Solvent C: NA Results Mass found is the mass found of the compoundspecification M/z found is the molecular ion found ((M + z)/z) of theand compound validation Calculated Mass is the molecular weight of thedesired compound Calculated M/z is the molecular weight (M + z)/z of thedesired compound

Method: LCMS13v01

System System: Waters Acquity UPLC SQD 2000 Column: Acquity UPLC BEH1.7μ C18 100 Å 2.1 × 100 mm Detector: UV: PDA, SQD 2000 DetectorIonisation method: ES+ setup Scanning range: 500-2000 Cone Voltage: 60 VScantime 0.5 Conditions Linear gradient: 10% to 90% B Gradient run-time:3 min Total run-time: 4 min Flow rate: 0.3 ml/min Column temperature:40° C. PDA: 210-400 nm Eluents Solvent A: 99.90% H2O, 0.1% TFA SolventB: 99.90% CH3CN, 0.1% TFA Solvent C: NA Results Mass found is the massfound of the compound specification M/z found is the molecular ion found((M + z)/z) of the and compound validation Calculated Mass is themolecular weight of the desired compound Calculated M/z is the molecularweight (M + z)/z of the desired compound

UPLC Methods Method: 04 A9 1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEH Shield RP18, C18, 1.7 um, 2.1 mm×150 mmcolumn, 60° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 200 mM Na2SO4+20 mM Na2HPO4+20 mM NaH2PO4 in 90% H2O/10% CH3CN, pH7.2;

B: 70% CH3CN, 30% H2O.

The following step gradient was used: 90% A, 10% B to 80% A, 20% B over3 minutes, 80% A, 20% B to 50% A, 50% B over 17 minutes at a flow-rateof 0.40 ml/min.

Method: 09 B2 1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1 mm×150 mm column,40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H2O, 0.05% TFA;

B: 99.95% CH3CN, 0.05% TFA.

The following linear gradient was used: 95% A, 5% B to 40% A, 60% B over16 minutes at a flow-rate of 0.40 ml/min.

Method: 09 B4 1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1 mm×150 mm column,40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H2O, 0.05% TFA;

B: 99.95% CH3CN, 0.05% TFA.

The following linear gradient was used: 95% A, 5% B to 5% A, 95% B over16 minutes at a flow-rate of 0.40 ml/min.

Method: 10 B4 1

The RP-analysis was performed using a Waters UPLC system fitted with adual band detector. UV detections at 214 nm and 254 nm were collectedusing an ACQUITY UPLC BEHC18, 1.7 um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H2O, 0.05% TFA;

B: 99.95% CH3CN, 0.05% TFA.

The following linear gradient was used: 95% A, 5% B to 5% A, 95% B over16 minutes at a flow-rate of 0.40 ml/min.

Method: UPLC01v01

System System: Waters Acquity UPLC system Column: ACQUITY UPLC BEH C18,1.7 um, 2.1 mm × 150 mm column Detectors: Waters Acquity TUV DetectorDetector 214 nm and 254 nm setup Conditions Linear gradient: 5% to 60% BGradient run-time: 16 minutes Total run-time: 20 minutes Flow rate: 0.40ml/min fixed Column temperature: 40° C. Eluents Solvent A: 99.95% Water,0.05% Trifluoroacetic acid Solvent B: 99.95% Acetonitrile, 0.05%Trifluoroacetic acid

Method: UPLC02v01

System System: Waters Acquity UPLC system Column: ACQUITY UPLC BEH C18,1.7 um, 2.1 mm × 150 mm column Detectors: Waters Acquity TUV DetectorDetector 214 nm and 254 nm setup Conditions Linear gradient: 5% to 95% BGradient run-time: 16 minutes Flow rate: 0.40 ml/min fixed Columntemperature: 40° C. Eluents Solvent A: 99.95% Water, 0.05%Trifluoroacetic acid Solvent B: 99.95% Acetonitrile, 0.05%Trifluoroacetic acid

Example 1N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28]-Glucagonamide

UPLC Method: 04_A9_1; Rt=15.8 min

LC-MS Method: LCMS_4; Rt=2.3 min; m/3: 1429; m/4: 1108; m/5: 887

UPLC Method: 10_B4_1; Rt=8.2 min

Example 2N^(ε21)-(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]aetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28]-Glucagonamide

UPLC Method: 10_B4_1; Rt=8.18 min

UPLC Method: 04_A9_1; Rt=17.4 min

LC-MS Method: LCMS_4; Rt=2.29; m/3:1468; m/4: 1101; m/5: 885

Example 3N^(ε24)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]-Glucagonamide

UPLC Method: 09_B4_1; Rt=8.4 min

UPLC Method: 04_A9_1; Rt=17.4 min

LC-MS Method: LCMS_4; Rt=2.4 min; m/3:1464; m/4: 1098; m/5: 879

Example 4N^(ε28)-(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Lys28]-Glucagonamide

UPLC Method: 10_B4_1; Rt=8.2 min

UPLC Method: 04_A9_1; Rt=16.2 min

LC-MS Method: LCMS_4; Rt=2.3 min; m/3:1477; m/4: 1108; m/5: 887

Example 5N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]aetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

UPLC Method: 10_B4_1; Rt=8.3 min

UPLC Method: 04_A9_1; Rt=15.2 min

LC-MS Method: LCMS_4; Rt=2.3 min; m/3:1473; m/4: 1105; m/5: 884

Example 6 N^(α)-([Aib2,Leu10,Arg20,Leu27,Ser28]-Glucagonyl)-N{Epsilon}[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]Lysamide

UPLC Method: 10_B4_1; Rt=8.0 min

UPLC Method: 04_A9_1; Rt=14.2 min

LC-MS Method: LCMS_4; Rt=2.3 min; m/4:1130; m/5: 904; m/6: 754

Example 7N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Glu21,Leu27]-Glucagonamide

UPLC Method: 10_B4_1; Rt=8.3 min

UPLC Method: 04_A9_1; Rt=15.3 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1472; m/4:1104; m/5:883

Example 8N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys7,Ala18,Glu21,Leu27,Lys29]-Glucagonamide

UPLC Method: 10_B4_1; Rt=8.9 min

UPLC Method: 04_A9_1; Rt=16.2 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1439; m/4: 1079; m/5: 863

Example 9N^(ε29)-[(4S)-4-carboxy-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

UPLC Method: 10_B4_1; Rt=8.6 min

UPLC Method: 04_A9_1; Rt=16.7 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1458; m/4: 1093; m/5: 875

Example 10N^(ε16)-[(4S)-4-carboxy-4-[[(4)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys6,Arg20,Glu21,Leu27]-Glucagonamide

UPLC Method: 10_B4_1; Rt=8.0 min

UPLC Method: 04_A9_1; Rt=16.6 min

LC-MS Method: LCMS_4; Rt=2.2 min, m/3: 1491; m/4: 1118; m/5: 895

Example 11N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.5 min

LC-MS Method: LCMS_4; Rt=2.28 min; m/6: 734

Example 12N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

UPLC Method: 09_B4_1; Rt=8.0 min

UPLC Method: 04_A9_1; Rt=14.7 min

LC-MS Method: LCMS_4; Rt=2.2 min, m/3: 1486; m/4: 1115; m/5: 892

Example 13N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.4 min

LC-MS Method: LCMS_4; Rt=2.2 min; m/6: 739

Example 14N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.5 min

UPLC Method: 04_A9_1; Rt=13.4 min

LC-MS Method: LCMS_4; Rt=2.3 min; m/3: 1108; m/4: 1006; m/5: 886

Example 15N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.4 min

UPLC Method: 04_A9_1; Rt=16.1 min

LC-MS Method: LCMS_4; Rt=2.3 min; m/3: 1477; m/4: 1108; m/5: 886

Example 16N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28]-Glucagonamide

UPLC Method: UPLC02v01: Rt=7.9 min

LC-MS Method: LCMS01v01: Rt=2.1 min; m/3: 1499; m/4: 1124; m/5: 900

Example 17N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagon amide

UPLC Method: 09_B4_1; Rt=8.5 min

UPLC Method: 04_A9_1; Rt=15.6 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1433; m/4: 1075; m/5: 860

Example 18N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

UPLC Method: 09_B4_1; Rt=8.4 min

UPLC Method: 04_A9_1; Rt=16.0 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1443; m/4: 1082; m/5: 866

Example 19N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

UPLC Method: UPLC01v01: Rt=12.2 min

LC-MS Method: LCMS01v01: Rt=1.9 min, m/3 1471; m/4 1103; m/5 882

Example 20N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanol]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: UPLC01v01: Rt=12.3 min

LC-MS Method: LCMS01v01:Rt=1.8 min, m/3 1485; m/4 1114; m/5 891

Example 21N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanol]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

UPLC Method: UPLC01v01: Rt=12.1 min

LC-MS Method: Rt=1.8 min, m/3 1490; m/4 1117; m/5 894

Example 22N^(ε29)-[(4S)-4-carboxy-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.5 min

UPLC Method: 04_A10_1; Rt=9.7 min

LC-MS Method: LCMS_4; Rt=2.6 min, m/3: 1476; m/4: 1107; m/5: 886

Example 23N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.5 min

UPLC Method: 04_A10_1; Rt=9.7 min

LC-MS Method: LCMS_4 Rt 2.6 m, m/3: 1486; m/4: 1114; m/5: 892

Example 24N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

UPLC Method: 09_B2_1; Rt=13.4 min

UPLC Method: 04_A10_1; Rt=10.1 min

LC-MS Method: LCMS_4; Rt=2.8 min, m/3: 1448; m/4: 1086; m/5: 869

Example 25N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.6 min

UPLC Method: 04_A9_1; Rt=12.2 min

LC-MS Method: LCMS_4; Rt=1.7 min, m/3: 1475; m/4: 1107; m/5: 885

Example 26N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]-acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

UPLC Method: 09_B2_1; Rt=13.3 min

UPLC Method: 04_A9_1; Rt=17.5 min

LC-MS Method: LCMS_4; Rt=1.8 min, m/3: 1481; m/4: 1111; m/5: 889

Example 27N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

UPLC Method: 09_B2_1; Rt=12.6 min

UPLC Method: 04_A9_1; Rt=12.7 min

LC-MS Method: LCMS_4; Rt=1.7 min, m/3: 1479; m/4: 1110; m/5: 888

Example 28N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29]-Glucagonamide

UPLC Method: UPLC02v01; Rt=7.7 min

LC-MS Method: LCMS13v01; Rt=2.1 min, m/3: 1461; m/4: 1096

Example 29N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Leu27]-Glucagonamide

UPLC Method: UPLC02v01; Rt=8.1 min

LC-MS Method: LCMS13v01; Rt=2.2 min, m/3: 1476; m/4: 1107

Example 30N^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.1 min

UPLC Method: 04_A9_1; Rt=14.3 min

LC-MS Method: LCMS_4; Rt=2.1 min, m/3: 1489; m/4: 1117; m/5: 893

Example 31N^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanol]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28]-Glucagon amide

UPLC Method: 09_B2_1; Rt=12.2 min

UPLC Method: 04_A9_1; Rt=14.2 min

LC-MS Method: LCMS_4; Rt=2.2 min, m/4: 1102; m/5: 882

Example 32N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28]-Glucagon amide

UPLC Method: 09_B2_1; Rt=12.1 min

UPLC Method: 04_A9_1; Rt=14.1 min

LC-MS Method: LCMS_4; Rt=2.1 min, m/4: 1102; m/5: 882

Example 33N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.2 min

UPLC Method: 04_A9_1; Rt=15.2 min

LC-MS Method: LCMS_4; Rt=2.0 min, m/3: 1489; m/4: 1117; m/5: 893

Example 34N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanol]-[Aib2,His3,Leu10Glu15,Lys17,Arg20,Glu2,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.2 min

UPLC Method: 04_A9_1; Rt=14.9 min

LC-MS Method: LCMS_4; Rt=2.0 min, m/3: 1383; m/4: 1037; m/5: 830

Example 35N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,His3,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

UPLC Method: UPLC02v01 Rt=8.14 min

LC-MS Method: LCMS01 v01; Rt=2.1; m/3:1478; m/5:1109; m/6:887

Example 36N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

UPLC Method: UPLC02v01: Rt=7.9 min

LC-MS Method: LCMS01v01: Rt=2.1; m/3: 1480; m/4: 1110; m/5: 888

Example 37N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,His3,Leu10Glu15,Arg20,Leu27,Lys28]-Glucagon amide

UPLC Method: UPLC02v01: Rt=7.8 min

LC-MS Method: LCMS01v01: Rt=2.1; m/3: 1484; m/4: 1113; m/5: 891

Example 38N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.8 min

UPLC Method: 04_A9_1; Rt=18.6 min

LC-MS Method: LCMS_4; Rt=2.9 min, m/3: 1446; m/4: 1084; m/5: 868

Example 39N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.3 min

UPLC Method: 04_A9_1; Rt=16.0 min

LC-MS Method: LCMS_4; Rt=2.8 min, m/3: 1432; m/4: 1074; m/5: 859

Example 40N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.2 min

UPLC Method: 04A9_1; Rt=13.9 min

LC-MS Method: LCMS_4; Rt=2.7 min, m/3: 1442; m/4: 1081; m/5: 865

Example 41N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

UPLC Method: 09_B2_1; Rt=12.3 min

UPLC Method: 04_A9_1; Rt=15.1 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1389; m/4: 1042; m/5: 834

Example 42N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.2 min

UPLC Method: 04_A9_1; Rt=14.7 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1486; m/4: 1114; m/5: 892

Example 43N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.4 min

UPLC Method: 04_A9_1; Rt=17.2 min

LC-MS Method: LCMS_4; Rt=2.4 min, m/3: 1476; m/4: 1107; m/5: 886

Example 44N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.3 min

UPLC Method: 04_A9_1; Rt=15.2 min

LC-MS Method: LCMS_4; Rt=2.3 min, m/3: 1490; m/4: 1117; m/5: 894

Example 45N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

UPLC Method: UPLC02v01 Rt=8.4 min

LC-MS Method: LCMS01v01; Rt=2.7 min; m/3:1532; m/5:1150; m/6:920

Example 46N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

UPLC Method: UPLC02v01 Rt=8.4 min

LC-MS Method: LCMS01v01; Rt=2.7 min; m/3:1542; m/5:1157; m/6:925

Example 47N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu2,Leu27,Lys28]-Glucagonamide

UPLC Method: UPLC02v01: Rt=8.4 min

LC-MS Method: LCMS01v01: Rt=2.7 min; m/3: 1528; m/4: 1146; m/5: 917

Example 48N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

UPLC Method: UPLC02v01: Rt=8.3 min

LC-MS Method: LCMS01v01: Rt=2.1 min; m/3: 1484; m/4: 1113; m/5: 891

Example 49N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.4 min

UPLC Method: 04_A9_1; Rt=14.5 min

LC-MS Method: LCMS_4; Rt=2.4 min, m/3: 1485; m/4: 1114; m/5: 891

Example 50N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC Method: 09_B2_1; Rt=12.4 min

UPLC Method: 04_A9_1; Rt=14.5 min

LC-MS Method: LCMS_4; Rt=2.4 min, m/3: 1485; m/4: 1114; m/5: 891

Example 51N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC01v01: Rt=12.7 min

LC-MS method: LCMS01 v01: Rt=2.7 min; m/3=1480; m/4=1110; m/5=888

Example 52N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: Rt=8.0 min

LC-MS method: LCMS01 v01: Rt=2.6 min; m/1=4570; m/3=1524; m/4=1143;m/5=915

Example 53N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.5 min

LC-MS method:LCMS01v01: Rt=2.8 min; m/1=4569; m/3=1523; m/4=1143;m/5=915

Example 54N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.0 min

LC-MS method:LCMS01 v01: Rt=2.5 min; m/3=1524; m/4=1143; m/5=915;m/z=4571

Example 55N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.4 min

LC-MS method:LCMS01 v01: Rt=2.8 min; m/3=1466; m/4=1100; m/5=880;m/z=4395

Example 56N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC01v01: RT=12.386 minLC-MS method: LCMS01v01: m/3=1481.4; m/4=1111.03; m/5=889.3

Example 57N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10Glu15,Arg20,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.0 min

LC-MS method: LCMS13v01: Rt=2.2 min; m/3=1480.8; m/4=1111.04

Example 58N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10Ala16Arg20,Leu27,Lys28]-Glucagon amide

UPLC method: UPLC02v01: RT=8.7 min

LC-MS method: LCMS13v01: Rt=2.2 min; m/3=1471.05; m/4=1103.69

Example 59N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.0 min

LC-MS method: LCMS01 v01: m/3=1542; m/4=1157; m/5=926; m/z=4626

Example 60N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=7.9 min

LC-MS method: LCMS01 v01: Rt=2.0 min; m/1=4587; m/3=1529; m/4=1147;m/5=918

Example 61N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.2 min

LC-MS method: LCMS01v01: Rt=2.1 min; m/1=4598; m/4=1150; m/5=920

Example 62N^(ε28)-[(4S)-4-carboxy-4-4-[[(4S)-4-carboxy-4-[[(4S)-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.2 min

LC-MS method: LCMS13v01: Rt=2.3 min; m/3=1384.04; m/4=1038.14

Example 63N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Acb2,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.3 min

LC-MS method: LCMS13v01: Rt=2.2 min; m/3=1476.15; m/4=1107.36

Example 64N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.3

LC-MS method: LCMS13v01: Rt=2.2 min; m/3=1471.27; m/4=1103.76

Example 65N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29]-Glucagonamide

UPLC method: UPLC02v01: RT=8.1 min

LC-MS method: LCMS01 v01: Rt=2.2 min; m/3=1476; m/4=1107; m/5=886

Example 66N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanol]-[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

UPLC method: UPLC02v01: RT=8.2 min

LC-MS method: LCMS01 v01: Rt=2.2 min; m/3=1467; m/4=1100; m/5=880

Example 67N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.0 min

LC-MS method: LCMS01 v01: Rt=2.1 min; m/3=1533; m/4=1150; m/5=920

Example 68N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=7.9 min

LC-MS method: LCMS01 v01: Rt=2.0 min; m/3=1528; m/4=1146; m/5=917

Example 69N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10Glu15,Lys17,Arg20,Ser21,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.1 min

LC-MS method: LCMS01 v01: Rt=2.6 min; m/3=1133; m/4=906; m/5=756

Example 70N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Ala16,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.4 min

LC-MS method: LCMS13v01: Rt=2.2 min; m/3=1457; m/4=1093; m/5=874

Example 71N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Leu16,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.5 min

LC-MS method: LCMS13v01: Rt=2.3 min; m/3=1471; m/4=1104

Example 72N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.4 min

LC-MS method: LCMS13v01: Rt=2.3 min; m/3=1467; m/4=1100

Example 73N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Leu16,Leu27,Lys28]-Glucagonamide

UPLC method: UPLC02v01: RT=8.5 min

LC-MS method: LCMS13v01: Rt=2.3 min; m/3=1481; m/4=1111

Example 74: GLP-1 and Glucagon Receptor Potency

The purpose of this example was to test the potency, of the glucagonderivatives of the invention, in vitro. The in vitro potency is themeasure of human GLP-1 receptor or glucagon receptor activation,respectively, in a whole cell assay.

Principle

In vitro potency was determined by measuring the response of human GLP-1or glucagon receptor, respectively, in a reporter gene assay. The assaywas performed in a stably transfected BHK cell line that expresseseither the human GLP-1 receptor or the human glucagon receptor andcontains the DNA for the cAMP response element (CRE) coupled to apromoter and the gene for firefly luciferase (CRE luciferase). When thehuman GLP-1 or glucagon receptor, respectively, was activated itresulted in the production of cAMP, which in turn resulted in theluciferase protein being expressed. When assay incubation was completed,the luciferase substrate (luciferin) was added and the enzyme convertedluciferin to oxyluciferin and produces bioluminescence. The luminescencewas measured as the readout for the assay.

(a) GLP-1 Receptor Activation Cell Culture and Preparation

The cells used in this assay (clone FCW467-12A/KZ10-1) were BHK cellswith BHKTS13 as a parent cell line. The cells were derived from a clone(FCW467-12A) that expresses the human GLP-1 receptor and wereestablished by further transfection with CRE luciferase to obtain thecurrent clone.

The cells were cultured at 5% CO₂ in DMEM medium with 10% FBS,1×GlutaMAX, 1 mg/ml G418, 240 nM MTX (methotrexate) and 1% pen/strep(penicillin/streptomycin). They were aliquoted and stored in liquidnitrogen. Before each assay, an aliquot was taken up and washed threetimes in PBS before being suspended at the desired concentration inassay medium. For 96-well plates the suspension was made to give a finalconcentration of 5×10EE3 cells/well.

Materials

The following chemicals were used in the assay: Pluronic F-68 (10%)(Gibco 2404), ovalbumin (Sigma A5503), DMEM w/o phenol red (Gibco11880-028), 1 M Hepes (Gibco 15630), Glutamax 100× (Gibco 35050) andsteadylite plus (PerkinElmer 6016757).

Assay Medium consisted of DMEM w/o phenol red, 10 mM Hepes, 1×GlutaMAX,2% Ovalbumin and 0.2% Pluronic F-68.

Procedure

Cell stocks were thawed in a 37° C. water bath. Cells were washed threetimes in PBS. The cells were counted and adjusted to 5×10EE3 cells/50 pl(1×10EE5 cells/ml) in Assay Medium. A 50 μl aliquot of cells wastransferred to each well in the assay plate.

Stocks of the test compounds and reference compounds were diluted to aconcentration of 0.2 pM in Assay Medium. Compounds were diluted 10-foldto give the following concentrations: 2×10EE-6 M, 2×10EE-7 M, 2×10EE-8M; 2×10EE-9 M, 2×10EE-10 M, 2×10EE-11 M, 2×10EE-12 M and 2×10EE-13 M.For each compound a blank assay medium control was also included.

A 50 μl aliquot of compound or blank was transferred from the dilutionplate to the assay plate. Compounds were tested at the following finalconcentrations: 1×10EE-6 M, 1×10EE-7 M, 1×10EE-8 M; 1×10EE-9 M,1×10EE-10 M, 1×10EE-11 M and 1×10EE-12 M and 1×10EE-13 M.

The assay plate was incubated for 3 h in a 5% CO₂ incubator at 37° C.The assay plate was removed from the incubator and allowed to stand atroom temperature for 15 min. A 100 μl aliquot of steadylite plus reagentwas added to each well of the assay plate (reagent is light sensitive).Each assay plate was covered with aluminum foil to protect it from lightand shaken for 30 min at room temperature. Each assay plate was read ina Perkin Elmer TopCount NXT instrument.

Calculations

The data from the TopCount instrument was transferred to GraphPad Prismsoftware. The software performed a non-linear regression (log(agonist)vs response-Variable slope (four parameter)). EC₅₀ values werecalculated by the software and reported in pM. Data are shown in Table6.

(b) Glucagon Receptor Activation Cell Culture and Preparation

The cells used in this assay (clone pLJ6′-4-25) were BHK cells withBHK570 as a parent cell line expressing the CRE luciferase gene (cloneBHK/KZ10-20-48) and were established by further transfection with thehuman glucagon receptor (clone pLJ6′ in pHZ-1 vector).

The cells were cultured at 5% CO₂ in DMEM medium with 10% FBS,1×GlutaMAX, 1 mg/ml G418, 240 nM MTX (methotrexate) and 1% pen/strep(penicillin/streptomycin). They were aliquoted and stored in liquidnitrogen. Before each assay, an aliquot was taken up and washed threetimes in PBS before being suspended at the desired concentration inassay medium. For 96-well plates the suspension was made to give a finalconcentration of 5×10EE3 cells/well.

Materials

The following chemicals were used in the assay: Pluronic F-68 (10%)(Gibco 2404), ovalbumin (Sigma A5503), DMEM w/o phenol red (Gibco11880-028), 1 M Hepes (Gibco 15630), Glutamax 100× (Gibco 35050) andsteadylite plus (PerkinElmer 6016757).

Assay Medium consisted of DMEM w/o phenol red, 10 mM Hepes, 1×GlutaMAX,2% Ovalbumin and 0.2% Pluronic F-68.

Procedure

Cell stocks were thawed in a 37° C. water bath. Cells were washed threetimes in PBS. The cells were counted and adjusted to 5×10EE3 cells/50 pl(1×10EE5 cells/ml) in Assay Medium. A 50 pl aliquot of cells wastransferred to each well in the assay plate.

Stocks of the test compounds and reference compounds were diluted to aconcentration of 0.2 pM in Assay Medium. Compounds were diluted 10-foldto give the following concentrations: 2×10EE-6 M, 2×10EE-7 M, 2×10EE-8M; 2×10EE-9 M, 2×10EE-10 M, 2×10EE-11 M, 2×10EE-12 M and 2×10EE-13 M.For each compound a blank assay medium control was also included.

A 50 μl aliquot of compound or blank was transferred from the dilutionplate to the assay plate. Compounds were tested at the following finalconcentrations: 1×10EE-6 M, 1×10EE-7 M, 1×10EE-8 M; 1×10EE-9 M,1×10EE-10 M, 1×10EE-11 M and 1×10EE-12 M and 1×10EE-13 M.

The assay plate was incubated for 3 h in a 5% CO₂ incubator at 37° C.The assay plate was removed from the incubator and allowed to stand atroom temperature for 15 min. A 100 μl aliquot of steadylite plus reagentwas added to each well of the assay plate (reagent is light sensitive).Each assay plate was covered with aluminum foil to protect it from lightand shaken for 30 min at room temperature. Each assay plate was read ina Perkin Elmer TopCount NXT instrument.

Calculations

The data from the TopCount instrument was transferred to GraphPad Prismsoftware. The software performed a non-linear regression (log(agonist)vs response-Variable slope (four parameter)). EC₅₀ values werecalculated by the software and reported in pM. Data are shown in Table6.

Example 75: GLP-1 and Glucagon Receptor Binding (a) GLP-1 ReceptorBinding

The purpose of this assay was to test the in vitro receptor bindingactivity of the glucagon derivatives of the invention. The receptorbinding is a measure of affinity of a compound for the human GLP-1receptor.

Principle

The receptor binding of each compound to the human GLP-1 receptor wasmeasured in a displacement binding assay. In this type of assay alabelled ligand (in this case 125I-GLP-1) is bound to the receptor. Eachderivative was added in a series of concentrations to isolated membranescontaining the human GLP-1 receptor and displacement of the labelledligand was monitored. The receptor binding was reported as theconcentration at which half of the labelled ligand was displaced fromthe receptor, the IC₅₀ value.

In order to test the binding of the derivatives to albumin, the assaymay be performed in a very low concentration of serum albumin (max.0.001% final assay concentration) as well as in the presence of a higherconcentration of serum albumin (2.0% final assay concentration). Anincrease of the IC₅₀ value, in the presence of serum albumin indicatesan affinity to serum albumin and represents a method to predict aprotracted pharmacokinetic profile of the test substance in animalmodels.

Materials

The following chemicals were used in the assay: DMEM w/o phenol red(Gibco 11880-028), Pen/strep (Invitrogen 15140-122), G418 (Invitrogen10131-027), 1 M Hepes (Gibco 15630), EDTA (Invitrogen 15575-038), PBS(Invitrogen 14190-094), fetal calf serum (Invitrogen 16140-071), EGTA,MgCl₂ (Merck 1.05832.1000), Tween 20 (Amresco 0850C335), SPA particles(wheat germ agglutinin (WGA) SPA beads, Perkin Elmer RPNQ0001),[125I]-GLP-1]-(7-36)NH2 (produced in-house), OptiPlate™-96 (PerkinElmer).

Buffer 1 consisted of 20 mM Na-HEPES plus 10 mM EDTA and pH was adjustedto 7.4.

Buffer 2 consisted of 20 mM Na-HEPES plus 0.1 mM EDTA and pH wasadjusted to 7.4.

Assay buffer consisted of 50 mM HEPES supplemented with 5 mM EGTA, 5 mMMgCl₂, 0.005% Tween 20 and pH was adjusted to 7.4.

Cell Culture and Membrane Preparation

The cells used in this assay (clone FCW467-12A) were BHK cells withBHKTS13 as a parent cell line. The cells express the human GLP-1receptor.

The cells were grown at 5% CO₂ in DMEM, 10% fetal calf serum, 1%Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the selectionmarker G418. To make a membrane preparation the cells were grown toapproximately 80% confluence. The cells were washed twice inphosphate-buffered saline and harvested. The cells were pelleted using abrief centrifugation and the cell pellet was kept on ice. The cellpellet was homogenised with ULTRA-THURRAX dispersing instrument for20-30 sec in a suitable amount of buffer 1 (e.g. 10 ml). The homogenatewas centrifuged for 15 min. The pellet was re-suspended (homogenised) in10 ml buffer 2 and centrifuged. This step was repeated once more. Theresulting pellet was re-suspended in buffer 2 and the proteinconcentration was determined. The membranes were aliquoted and stored atminus 80° C.

Procedure

1. For the receptor binding assay, 50 μl of the assay buffer was addedto each well of an assay plate.

2. Test compounds were serially diluted to give the followingconcentrations: 8×10EE-7 M, 8×10EE-8 M, 8×10EE-9 M, 8×10EE-10 M,8×10EE-11 M, 8×10EE-12 M and 8×10EE-13 M. Twenty-five pl were added toappropriate wells in the assay plate.

3. Cell membrane aliquots were thawed and diluted to their workingconcentration. Fifty μl were added to each well in the assay plate.

4. WGA SPA beads were suspended in assay buffer at 20 mg/ml. Thesuspension was diluted to 10 mg/ml in assay buffer just prior toaddition to the assay plate. Fifty pl were added to each well in theassay plate.

5. The incubation was started by adding 25 μl of 480 pM solution of[125I]-GLP-1]-(7-36)NH₂ to each well of the assay plate. A 25 μl aliquotwas reserved for measuring total counts/well.

6. The assay plate was incubated for 2 h at 30° C.

7. The assay plate was centrifuged for 10 min.

8. The assay plate was read in a Perkin Elmer TopCount NXT instrument.

Calculations

The data from the TopCount instrument were transferred to GraphPad Prismsoftware. The software averaged the values for the replicates andperformed a non-linear regression. IC₅₀ values were calculated by thesoftware and reported in nM. Data are shown in Table 6.

(b) Glucagon Receptor Binding

The purpose of this assay was to test the in vitro receptor bindingactivity (i.e. affinity) of the glucagon derivatives of the invention.The receptor binding activity is a measure of affinity of a derivativefor the human glucagon receptor.

Principle

The receptor binding of each compound to the human glucagon receptor wasmeasured in a displacement binding assay. In this type of assay alabelled ligand (in this case 125I-glucagon) is bound to the receptor.Each derivative was added in a series of concentrations to isolatedmembranes containing the human glucagon receptor and displacement of thelabelled ligand is monitored. The receptor binding was reported as theconcentration at which half of the labelled ligand is displaced from thereceptor, the IC₅₀ value.

In order to test the binding of the derivatives to albumin, the assaymay be performed in a very low concentration of serum albumin (max.0.001% final assay concentration) as well as in the presence of a higherconcentration of serum albumin (0.2% final assay concentration). Anincrease of the IC₅₀ value, in the presence of serum albumin indicatesan affinity to serum albumin and represents a method to predict aprotracted pharmacokinetic profile of the test substance in animalmodels.

Materials

The following chemicals were used in the assay: DMEM w Glutamax (Gibco61965-026), Pen/strep (Invitrogen 15140-122), G418 (Invitrogen10131-027), Versene (Gibco 15040), 1 M Hepes (Gibco 15630), PBS(Invitrogen 14190-094), fetal calf serum (Invitrogen 16140-071), MgCl₂(Merck 1.05832.1000), EDTA (Invitrogen 15575-038), CaCl₂ (Sigma, C5080),Tween 20 (Amresco 0850C335), ovalbumin (Sigma A5503), SPA particles(wheat germ agglutinin (WGA) SPA beads, Perkin Elmer RPNQ0001),[125I]-glucagon (produced in-house), OptiPlate™-96 (Packard 6005290).

HME buffer consisted of 25 mM HEPES, 2 mM MgCl₂ and 1 mM EDTA, and pHwas adjusted to 7.4. Assay buffer consisted of 50 mM HEPES supplementedwith 5 mM MgCl₂, 1 mM CaCl₂, 0.02% Tween 20 and 0.1% Ovalbumin, and pHwas adjusted to 7.4.

Cell Culture and Membrane Preparation

The cells used in this assay (clone BHK hGCGR A3*25) were BHK cellsstable transfected with an expression plasmid containing the cDNAencoding the human glucagon receptor.

The cells were grown at 5% CO₂ in DMEM, 10% fetal calf serum, 1%Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the selectionmarker G418. To make a membrane preparation the cells were grown toapproximately 80% confluence. The cells were washed twice inphosphate-buffered saline and harvested. The cells were pelleted using abrief centrifugation and the cell pellet was kept on ice. Lyse the cellsby adding approx. 5 ml HME buffer, mix by pipetting and snap freeze inliquid nitrogen. Thaw quickly and add HME buffer to 10 ml. The cellpellet was homogenised with an ULTRA-THURRAX dispersing instrument for20-30 sec. The homogenate was centrifuged at 20.000×G, 4° C. for 10 min.The pellet was re-suspended (homogenised) in 1-2 ml HME buffer. Theprotein concentration was determined. The membranes were aliquoted andsnap-frozen in liquid nitrogen and stored at minus 80° C.

Procedure

1. For the receptor binding assay, 50 pl of the assay buffer was addedto each well of an assay plate.

2. Test compounds were serially diluted to give the followingconcentrations: 8×10EE-7 M, 8×10EE-8 M, 8×10EE-9 M, 8×10EE-10 M,8×10EE-11 M, 8×10EE-12 M and 8×10EE-13 M. Twenty-five pl were added toappropriate wells in the assay plate.

3. Cell membrane aliquots were thawed and diluted to their workingconcentration. Fifty μl were added to each well in the assay plate.

4. WGA SPA beads were suspended in assay buffer at 20 mg/ml. Thesuspension was diluted to 10 mg/ml in assay buffer just prior toaddition to the assay plate. Fifty pl were added to each well in theassay plate.

5. The incubation was started by adding 25 pl of 480 pM solution of[125I]-glucagon to each well of the assay plate. A 25 μl aliquot wasreserved for measuring total counts/well.

6. The assay plate was incubated for 2 h at 25° C.

7. The assay plate was centrifuged for 10 min at 1500 rpm.

8. The assay plate was read in a Perkin Elmer TopCount NXT instrument.

Calculations

The data from the TopCount instrument were transferred to GraphPad Prismsoftware. The software averaged the values for the replicates andperformed a non-linear regression. IC₅₀ values were calculated by thesoftware and reported in nM. Data are shown in Table 6.

Example 76: Thioflavin T (ThT) Fibrillation Assay for the Assessment ofPhysical Stability of Peptide Formulations

The purpose of this assay was to assess the physical stability of theglucagon derivatives of the invention in aqueous solutions.

Low physical stability of a peptide may lead to amyloid fibrilformation, which is observed as well-ordered, thread-like macromolecularstructures in the sample, which eventually may lead to gel formation.This has traditionally been measured by visual inspection of the sample.However, that kind of measurement is very subjective and depending onthe observer. Therefore, the application of a small molecule indicatorprobe is much more advantageous. Thioflavin T (ThT) is such a probe andhas a distinct fluorescence signature when binding to fibrils [Naiki etal; Anal. Biochem. 1989 177 244-249; LeVine; Methods. Enzymol. 1999 309274-284].

The time course for fibril formation can be described by a sigmoidalcurve with the following expression [Nielsen et al; Biochemistry 2001 406036-6046]:

$\begin{matrix}{F = {f_{i} + {m_{i}t} + \frac{f_{f} + {m_{f}t}}{1 + e^{- {\lbrack{{({t - t_{0}})}/\tau}\rbrack}}}}} & {{Eq}.\mspace{14mu} (1)}\end{matrix}$

Here, as depicted in FIG. 1, F is the ThT fluorescence at the time t.The constant t₀ is the time needed to reach 50% of maximum fluorescence.The two important parameters describing fibril formation are thelag-time calculated by t₀−2τ and the apparent rate constant kapp 1/τ.

Formation of a partially folded intermediate of the peptide is suggestedas a general initiating mechanism for fibrillation. Few of thoseintermediates nucleate to form a template onto which furtherintermediates may assembly and the fibrillation proceeds. The lag-timecorresponds to the interval in which the critical mass of nucleus isbuilt up and the apparent rate constant is the rate with which thefibril itself is formed.

Samples were prepared freshly before each assay. Each sample compositionis described in the legends. The pH of the sample was adjusted to thedesired value using appropriate amounts of concentrated NaOH and HCl.Thioflavin T was added to the samples from a stock solution in H₂O to afinal concentration of 1 μM.

Sample aliquots of 200 μl (250 pM of the glucagon derivative/analogue in10 mM HEPES buffer, pH 7.5) were placed in a 96 well microtiter plate(Packard OptiPlate™-96, white polystyrene). Usually, four or eightreplica of each sample (corresponding to one test condition) were placedin one column of wells. The plate was sealed with Scotch Pad (Qiagen).

Incubation at given temperature, shaking and measurement of the ThTfluorescence emission were done in a Fluoroskan Ascent FL fluorescenceplate reader (Thermo Labsystems). The temperature was set to 37° C. andthe plate was incubated with orbital shaking adjusted to 960 rpm with anamplitude of 1 mm. Fluorescence measurement was done using excitationthrough a 444 nm filter and measurement of emission through a 485 nmfilter.

Each run was initiated by incubating the plate at the assay temperaturefor 10 min. The plate was measured every 20 minutes for a desired periodof time. Between each measurement, the plate was shaken and heated asdescribed.

After completion of the ThT assay the four or eight replica of eachsample was pooled and centrifuged at 20000 rpm for 30 minutes at 18° C.The supernatant was filtered through a 0.22 μm filter and an aliquot wastransferred to a HPLC vial.

The concentration of peptide in the initial sample and in the filteredsupernatant was determined by reverse phase HPLC using an appropriatestandard as reference. The percentage fraction the concentration of thefiltered sample constituted of the initial sample concentration wasreported as the recovery.

The measurement points were saved in Microsoft Excel format for furtherprocessing and curve drawing and fitting was performed using GraphPadPrism. The background emission from ThT in the absence of fibrils wasnegligible. The data points were typically a mean of four or eightsamples.

The data set may be fitted to Eq. (1). However, the lag time beforefibrillation may be assessed by visual inspection of the curveidentifying the time point at which ThT fluorescence increasessignificantly above the background level. The data, as shown in Table 6,strongly confirmed the improved physical stability of the compounds ofthis invention.

Example 77: In Vivo Test in Diet-Induced Obese (DIO)-Mice

The purpose of this experiment was to investigate effect of theglucagon-GLP-1R co-agonists from Example 8 and Example 12 on body weightand blood glucose in vivo in DIO mice. Liraglutide (a human GLP-1analogue marketed by Novo Nordisk A/S under the brand name Victoza™) wasincluded for comparison.

Male c57bl/6J mice obtained from Jackson Lab (USA) were fed a high fatdiet (Research Diets D12451, 45% kcal fat) to obtain approximately 50%overweight compared to age-matched lean control mice. The DIO mice arecharacterized by normoglycemia, insulin resistance and obesity. DIO micewith a body weight of approximately 40 g were allocated to groups of 8animals to obtain comparable body weight and body fat proportion betweengroups.

The co-agonists, i.e. the glucagon derivatives, were dissolved in 50 mMNa₂HPO₄, 145 mM NaCl og 0.05% Tween 80, pH 7.4, while liraglutide wasdiluted from 6 mg/ml Victoza™ pens in DPBS buffer (Invitrogen)containing 0.5% rat serum albumin. Vehicle treated animals received 50mM Na₂HPO₄, 145 mM NaCl og 0.05% Tween 80, pH 7.4. Compounds and vehiclewere dosed (1 ml/kg) for 5 weeks s.c. once daily just prior to lightsout. For the first half of the study co-agonists were administered at adose of 5 nmol/kg and for the second half of the study co-agonists wereadministered at a dose of 10 nmol/kg. For comparison, liraglutide at adose of 100 nmol/kg, likewise dosed s.c. once daily, was included. Bodyweight was monitored before dosing, daily during the dosing period.Non-fasted blood glucose was measured on the day of initiation of dosingand weekly during the dosing period, prior to dosing. Blood formeasurement of blood glucose was taken from the tail tip capillary andblood glucose was measured using a glucose analyzer (Biosen 5040) basedon the glucose oxidase method.

The body weight reduction/body weight at the end of the 5 week treatmentperiod and area under the blood glucose curve during the five weektreatment period are given in the tables below.

TABLE 1 Normalized body weight after 5 weeks of treatment Percent BW ofbaseline at Compound week 5 (mean ± SD) Body weight Vehicle 103% ± 2% 42 g ± 4 g  Liraglutide 87% ± 4% 36 g ± 5 g* Example 8 88% ± 8% 36 g ± 5g* Example 12 75% ± 8%  31 g ± 2 g** *p < 0.05; **p < 0.001 vs vehicle,One-way ANOVA, Bonferoni post-hoc test

TABLE 2 Area under the blood glucose curve measured during the 5-weektreatment period Compound AUC BG_((week 1-5)) (mean ± SD) Vehicle 243mM*day ± 16 mM*day   Liraglutide 208 mM*day ± 10 mM*day*** Example 8 187mM*day ± 11 mM*day*** Example 12 206 mM*day ± 16 mM*day*** ***p < 0.001vs vehicle, One-way ANOVA, Bonferoni post-hoc test

The results in Tables 1 and 2 show that the glucagon-GLP-1R co-agonistderivatives of the invention are biologically active in vivo, witheffective lowering of body weight, which for the compound from Example12 tends to be superior to liraglutide alone, while blood glucoseremains improved as compared to vehicle treated animals and on par withliraglutide treated animals.

Example 78: Pharmacokinetic Profile in Mice

The purpose of this assay was to assess the pharmocokinetic profiles ofthe glucagon derivatives of the invention in mice.

The pharmacokinetic profile of glucagon-GLP1 co-agonists was tested innormal male c57/BL6 mice (approximately body weight: 30 grams), in asparse sampling design with for example 3 mice represented at each timepoint (for example: t=0.25, 0.5, 1, 3, 6, 10, 24, 30 and 48 hours). Thecompound was dosed as a single subcutaneous dose of 10 nmol/kg at t=0.

The plasma levels of the glucagon-GLP1 co-agonist compounds weredetermined using an ELISA/LOCI assay (see Example 80 herein).Pharmacokinetic calculations such as half-life (T_(1/2)) maximumconcentration (C_(max)) and time for maximum concentration (T_(max)) ofthe compounds were determined using the PC based software, Phoenix(WinNonLin version 6.3 from Pharsight, Certara).

The results strongly confirmed the protracted effect of theglucagon-GLP1 co-agonists of this invention.

TABLE 3 Mean half-life (T_(1/2)), maximum concentration (C_(max)) andtime for maximum concentration (T_(max)) Compound T_(1/2) (hours)C_(max) (pM) T_(max) (hours) Example 8 23 45,000 3 Example 12 14 24,4003

Example 79: Chemical Stability Assessment

The purpose of this assay was to assess the chemical stability of theglucagon derivatives of the invention in aqueous solutions.

Chemical stability of glucagon derivatives or analogues was investigatedby RP-UPLC separation and UV detection. Lyophilized samples weredissolved in a 8 mM Phosphate buffer pH 8.6, followed by adjustment topH 7.3.using HCl to a final concentration of 333 pM. Samples wereincubated for 14 days at 5° C. and 37° C. followed by RP-UPLC analysis.Purity was defined as the area percentage of the main peak in relationto the total area of all integrated peaks in each chromatogram. Purityloss after 14 days at 37° C. was determined as the difference in puritybetween the samples incubated at 5° C. and 37° C., divided by the purityof the sample after incubation for 14 days at 5° C.

RP-UPLC analysis was performed using a Waters BEH130 2.1 mm×150 mm, 1.7μm column operated at 50° C. and a flow rate of 0.4 mL/min using amobile phase system consisting of A: 0.05% TFA in MQ-water B: 0.05% TFAin Acetonitrile. UV-detection was performed at 215 nm. The typicalgradient profile used for most of the samples is shown below.

TABLE 4 Typical gradient profile used for RP-UPLC analysis Time (min) %B Injection 20 30 60 31 99 37 99 39 20 40 20 45 20

For some individual derivatives or analogues eluting at substantiallydifferent retention times compared with the majority of derivatives oranalogues, some adjustments to the gradient profile were made to betterenable purity assessment comparison across samples. The data confirmedthe improved chemical stability of the glucagon derivatives of thisinvention.

TABLE 5 Chemical stability of GLP-1/glucagon receptor co-agonists Datashown are purity loss after 14 days at 37° C. in percent (%) Purityloss; Compound 14 days at 37° C. Example 8 9 Example 12 8 Example 16 3Example 22 1 Example 23 2 Example 25 1 Example 26 2 Example 27 5 Example34 5 Example 36 2 Example 38 2 Example 39 3 Example 40 2 Example 41 1Example 42 3 Example 43 2 Example 44 2 Example 45 3 Example 46 1 Example47 3 Example 48 2 Example 49 2 Example 51 3 Example 54 1 Example 55 2Example 57 1 Example 58 2 Example 60 4 Example 56 4 Example 62 2 Example63 1 Example 65 3 Example 66 2 Example 68 3 Example 67 4 Example 69 2Example 70 2

Example 80: ELISA/LOCI Assay for Determination of Peptides in BloodPlasma

The purpose of this assay was to determine the content of GLP-1/glucagonreceptor co-agonists in blood plasma.

Samples were analysed for peptide levels using Luminescence OxygenChanneling Immunoassay (LOCI). The donor beads were coated withstreptavidin. Acceptor beads are conjugated with a monoclonal antibodyspecific to gGlu in the linker (NN454-1F31) while the second monoclonalantibody (1F120) specific for glucagon was biotinylated. Three reactantswere combined with the analyte and formed a two-sited immuno-complex.Illumination of the complex released singlet oxygen atoms from the donorbeads. They were channeled into the acceptor beads and triggeredchemiluminescence which was measured in the EnVision plate reader. Theamount of emitted light is proportional to the concentration of peptide.

One μL sample/calibrator/control was applied to the wells of 384-wellLOCI plates followed by a 15 μL mixture of the antibody-coated acceptorbeads (0.5 μg/well) and the biotinylated antibody. The plates wereincubated over night at 21-22° C. Following incubation, 30 μL of thestreptavidin-coated donor-beads (2 μg/well) was added to each well andincubated for 30 minutes at 21-22° C. The plates were read in anEnvision plate reader at 21-22° C. with a filter having a bandwidth of520-645 nm after excitation by a 680 nm laser. The total measurementtime per well was 210 ms including a 70 ms excitation time.

The following table shows data of the glucagon derivatives of theinvention obtained as described in the examples above.

TABLE 6 EC₅₀ (Assay described in Example 74 herein) and IC₅₀ (Assaydescribed in Example 75 herein) values for GLP-1/glucagon receptorco-agonists and physical stability data (Assay described in Example 76herein) Com- EC₅₀ EC₅₀ IC₅₀ IC₅₀ pound of GLP-1R GlucR GLP-1R GlucR THTassay THT assay Example [pM] [pM] [nM] [nM] Lag time Recovery No. (Ex.74) (Ex. 74) (Ex. 75) (Ex. 75) (Ex. 76) (Ex. 76) 1 8.0 54.0 45.0 100.0 244.0 97.0 7.0 2.0 3 6.0 60.0 45.0 100.0 4 4.0 16.0 45.0 100.0 5 5.0109.0 45.0 100.0 6 20.0 1210.0 45.0 100.0 7 2.4 1.1 45.0 100.0 8 29.61128.1 1.1 6.7 45.0 98.0 9 3.2 698.9 .2 380.8 8.0 45.0 10 9.2 42.3 .51.2 7.0 11.0 11 15.0 672.0 12 14.7 104.0 .5 7.0 45.0 100.0 13 6.0 1430.014 28.0 599.0 15 20.0 190.0 16 5.8 113.1 .2 3.9 45.0 100.0 17 .2 53.045.0 92.6 18 2.3 220.3 .1 11.0 45.0 103.3 19 4.7 39.9 .9 8.5 6.7 5.0 204.4 62.4 .4 6.7 6.0 5.0 21 2.7 240.3 .3 51.0 18.0 33.0 22 11.8 87.1 .27.1 45.0 100.0 23 22.1 77.3 .3 6.4 45.0 104.0 24 11.9 506.3 .2 61.0 28.0105.0 25 5.7 44.7 .2 5.1 45.0 104.0 26 9.0 8.7 .5 4.0 45.0 100.0 27 4.11.7 .9 .7 42.0 98.0 28 29.5 1328.0 1.6 148.0 39 26.6 58.8 1.4 5.9 33.077.0 30 35.7 4.8 1.4 .5 45.0 100 31 19.5 2.9 1.5 .8 45.0 100 32 32.7 6.83.9 1.9 45.0 100 33 14.6 3.6 .6 2.0 45 100 34 14.7 2.9 .2 .2 45 100 353.0 1.0 2.6 .7 45 119 36 2.0 4.0 .2 .6 45 131 37 4.0 2.0 .2 .1 45 50 3813.0 24.0 1.2 29.4 45 99 39 4.0 12.0 .3 5.6 45 98 40 4.0 136.0 .4 37.740 96 41 7.0 29.0 .3 1.2 45 100 42 3.0 32.0 .3 38.2 45 100 43 5.0 2.0 .35.6 45 105 44 3.0 10.0 .3 7.2 45 105 45 18.0 6.0 .4 4.5 45 105 46 21.016.0 1.1 8.5 45 105 47 17.0 5.0 .2 1.5 45 105 48 44.0 8.0 .2 3.1 45 10549 17.0 4.0 .4 1.7 45 104 50 11.0 280.0 2.2 26.2 28 80 51 6.0 4.0 .7 1.445 104 52 4.0 16.0 .2 45 88 53 11.0 7.0 1.9 45 100 54 2.7 12.7 .2 1.7 45100 55 27.0 30.5 1.1 18.4 45 100 56 5.3 6.3 .3 2.1 45 100 57 3.0 129.0.2 19.0 45 100 58 4.3 11.3 .2 2.6 45 100 59 4.0 17.0 .2 1.4 12 9 60 6.054.0 .2 4.0 45 100 61 6.0 11.0 .3 1.8 20 27 62 8.0 10.0 .2 3.9 45 100 637.0 24.0 .8 7.7 45 100 64 3.0 20.0 .9 5.5 19 27 65 6.0 44.0 .5 17.0 45100 66 5.0 47.0 .6 10.4 45 104 67 4.0 10.0 .3 1.5 45 100 68 7.0 5.0 .3.6 45 100 69 7.0 68.0 0.2 9.5 45 105 70 1.5 30.0 0.2 3.2 45 106 71 4.017.0 0.7 5.7 45 100 72 3.0 57.0 0.5 11.3 31 98 73 5.0 49.0 1.5 16.2 2288

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A glucagon derivative comprising: a peptide comprising the amino acidsequenceHis-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₁₇-X₁₈-Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀;a substituent comprising a lipophilic moiety and at least threenegatively charged moieties; and a C-terminal amide; wherein, X₂represents Aib, Acb or Acpr; X₃ represents Gln or His; X₁₀ representsLeu, Ile or Val; X₁₂ represents Lys or Arg; X₁₅ represents Asp or Glu;X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys; X₁₇represents Arg or Lys; X₁₈ represents Arg, Ala or Lys; X₂₀ representsGln, Arg, Glu, Aib or Lys; X₂₁ represents Asp, Glu, Ser, or Lys; X₂₄represents Gln, Ala, Arg, Glu, Aib or Lys; X₂₇ represents Met, Leu orVal; X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys; X₂₉represents Thr, Gly, Ser, Gln, Ala, Glu or Lys; and X₃₀ represents Lysor is absent; wherein Lys is present at one or more positions selectedfrom the group consisting of X₁₂, X₁₆, X₁₇, X₁₈, X₂₀, X₂₁, X₂₄, X₂₈,X₂₉, and X₃₀ wherein said substituent comprisesZ₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-; wherein Z₁ is

n is 6-20, and the symbol * represents the attachment point to thenitrogen of a neighbouring group; wherein Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈,Z₉, and Z₁₀ individually are absent or an amino acid selected from thegroup consisting of Glu, γGlu, Gly, Ser, Ala, Thr, and Ado; wherein atleast two of residues Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, and Z₁₀ arepresent; wherein Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀ together contains atleast three negative charges; wherein one of said negatively chargedmoieties is distal of said lipophilic moiety; and wherein saidsubstituent is attached to said peptide at the epsilon position of a Lysresidue at one amino acid position selected from the group consisting ofX₁₆, X₁₇, X₁₈, X₂₀, X₂₁, X₂₄, X₂₈, X₂₉, and X₃₀; or a pharmaceuticallyacceptable salt or prodrug thereof.
 2. The glucagon derivative accordingto claim 1, wherein X₁₀ represents Leu; X₁₆ represents Ser, Ala, Leu,Thr, Glu, Aib, or Lys; X₁₈ represents Arg or Ala; X₂₀ represents Gln,Arg, Glu, or Lys; X₂₁ represents Asp, Glu, or Lys; X₂₄ represents Gln,Ala, Arg, or Lys; X₂₈ represents Asn, Ser, or Lys; and X₂₉ representsThr, Gly, or Lys.
 3. The glucagon derivative according to claim 1,wherein X₁₀ represents Leu.
 4. The glucagon derivative according toclaim 1, wherein said peptide comprises 3-15 amino acid residuemodifications SEQ ID NO:
 1. 5. The glucagon derivative according toclaim 1, wherein said substituent is selected from the group consistingof

wherein * represents the point of attachment to said peptide at thenitrogen atom of the epsilon position of a Lys residue.
 6. The glucagonderivative according to claim 1 wherein the glucagon derivative isselected from the group consisting of:N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys6,Arg20,Leu27,Ser28]-Glucagonamide

N^(ε21)-(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28]-Glucagonamide

N^(ε24)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

N^(α)-([Aib2,Leu10,Arg20,Leu27,Ser28]-Glucagonyl)-N{Epsilon}[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]Lysamide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Glu21,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]aetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagon amide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]-acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29]-Glucagonamide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Leu27]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28]-Glucagon amide

N^(ε16)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28]-Glucagon amide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,His3,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagon amide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu5,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu1010,Arg12,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu1010,Aib16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Acb2,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29]-GlucagonamideN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Ser21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Ala16,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Leu16,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

andN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Leu16,Leu27,Lys28]-Glucagonamide


7. A glucagon peptide consisting of (i) a C-terminal amide and (ii) theamino acid sequence of SEQ ID NO: 1 modified by a set of modificationsselected from the group consisting of: a)[Aib2,Leu10,Lys16,Arg20,Leu27,Ser28]; b)[Aib2,Leu10,Arg20,Lys21,Leu27,Ser28]; c)[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]; d) [Aib2,Leu10,Arg20,Leu27,Lys28];e) [Aib2,Leu10,Arg20,Leu27,Ser28,Lys29]; f)[Aib2,Leu10,Arg20,Leu27,Ser28]; g) [Aib2,Leu10,Lys16,Lys17,Glu21,Leu27];h) [Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29]; i)[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29]; j)[Aib2,Leu10,Lys16,Arg20,Glu21,Leu27]; k)[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29]; l)[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29]; m)Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29]; n)[Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29]; o)[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29]; p)[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28]; q)[Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27]; r)[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27]; s)[Aib2,Leu10,Lys16,Arg20,Glu21,Ala24,Leu27]; t)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; u)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]; v)[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; w)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; x)[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28]; y)[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; z)[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]; aa)[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; bb)[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29]; cc)[Aib2,Leu10,Lys16,Glu21,Leu27]; dd)[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; ee)[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28]; ff)[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28]; gg)[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; hh)[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; ii)[Acb2,His3,Leu10,Glu15,Leu27,Lys28]; jj)[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28] kk)[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28]; ll)[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29]; mm)[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29]; nn)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]; oo)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; pp)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; qq)[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28]; rr)[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; ss)[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]; tt)[Acb2,Leu10,Arg12,Leu16,Arg20,Leu27,Lys28]; uu)[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]; vv)[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]; ww)[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28]; xx)[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; yy)[Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28]; zz)[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; aaa)[Aib2,Leu10,Leu16,Leu27,Lys28]; bbb)[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; ccc)[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28]; ddd)[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28]; eee)[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28];fff)[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]; ggg)[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]; hhh)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; iii)[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; jjj)[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28]; kkk)[Acb2,Leu10,Glu15,Leu27,Lys28]; lll)[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28]; mmm)[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29]; nnn)[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]; ooo)[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]; ppp)[Aib2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]; qqq)[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]; rrr)[Aib2,Leu10,Glu15,Lys17,Arg20,Ser21,Leu27,Lys28]; sss)[Aib2,Val10,Ala16,Leu27,Lys28]; ttt) [Aib2, Val10,Leu16,Leu27,Lys28];uuu) [Aib2,Val10,Arg12,Ala16,Leu27,Lys28]; vvv)[Aib2,Val10,Arg12,Leu16,Leu27,Lys28]; or a pharmaceutically acceptablesalt, amide, or ester thereof.
 8. The glucagon derivative according toclaim 1, wherein the substituent is attached to said peptide at theepsilon position of a Lys residue at a position selected from the groupconsisting of X₂₄, X₂₈, X₂₉, and X₃₀.
 9. The glucagon derivativeaccording to claim 1, wherein the glucagon derivative is an agonist of aGLP-1 receptor with an EC50<100 pM and an agonist of a glucagon receptorwith an EC50<1 nM.
 10. The glucagon derivative according to claim 1,wherein the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide


11. The glucagon derivative according to claim 1, wherein the glucagonderivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide


12. The glucagon derivative according to claim 1, wherein the glucagonderivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide


13. The glucagon derivative according to claim 1, wherein the glucagonderivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide


14. The glucagon derivative according to claim 1, wherein the glucagonderivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-Glucagonamide


15. A pharmaceutical composition comprising (i) a glucagon derivativeand (ii) a GLP-1 compound or an insulin compound; wherein the glucagonderivative comprises a peptide comprising the amino acid sequenceHis-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₁₇-X₁₈-Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀;a substituent comprising a lipophilic moiety and at least threenegatively charged moieties; and a C-terminal amide; wherein, X₂represents Aib, Acb or Acpr; X₃ represents Gln or His; X₁₀ representsLeu, Ile or Val; X₁₂ represents Lys or Arg; X₁₅ represents Asp or Glu;X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys; X₁₇represents Arg or Lys; X₁₈ represents Arg, Ala or Lys; X₂₀ representsGln, Arg, Glu, Aib or Lys; X₂₁ represents Asp, Glu, Ser, or Lys; X₂₄represents Gln, Ala, Arg, Glu, Aib or Lys; X₂₇ represents Met, Leu orVal; X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys; X₂₉represents Thr, Gly, Ser, Gln, Ala, Glu or Lys; and X₃₀ represents Lysor is absent; wherein Lys is present at one or more positions selectedfrom the group consisting of X₁₂, X₁₆, X₁₇, X₁₈, X₂₀, X₂₁, X₂₄, X₂₈,X₂₉, and X₃₀; wherein said substituent comprisesZ₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-; wherein Z₁ is

n is 6-20, and the symbol * represents the attachment point to thenitrogen of a neighbouring group; wherein Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈,Z₉, and Z₁₀ individually are absent or an amino acid selected from thegroup consisting of Glu, γGlu, Gly, Ser, Ala, Thr, and Ado; wherein atleast two of residues Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, and Z₁₀ arepresent; wherein Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀ together contains atleast three negative charges; wherein one of said negatively chargedmoieties is distal of said lipophilic moiety; and wherein saidsubstituent is attached to said peptide at the epsilon position of a Lysresidue at one amino acid position selected from the group consisting ofX₁₆, X₁₇, X₁₈, X₂₀, X₂₁, X₂₄, X₂₈, X₂₉, and X₃₀; or a pharmaceuticallyacceptable salt or prodrug thereof.
 16. The pharmaceutical compositionaccording to claim 15, wherein the glucagon derivative is selected fromthe group consisting of:N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

andN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-Glucagonamide


17. The pharmaceutical composition according to claim 15, wherein theGLP-1 compound is selected from the group consisting of:N-epsilon26-((S)-4-Carboxy-4-hexadecanoylamino-butyryl)[Arg34]GLP-1-(7-37)

and N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)

and their pharmaceutically acceptable salts, amides, alkyls, or esters.18. The pharmaceutical composition according to claim 15, wherein theinsulin compound isN-epsilon-B29-[(S)-4-Carboxy-4-(15-carboxypentadecanoylamino)butyryl]desB30human insulin


19. A method of treating a disease or condition by administering to apatient in need thereof a glucagon derivative, or a pharmaceuticallyacceptable salt or prodrug thereof, comprising: a peptide comprising theamino acid sequenceHis-X₂-X₃-Gly-Thr-Phe-Thr-Ser-Asp-X₁₀-Ser-X₁₂-Tyr-Leu-X₁₅-X₁₆-X₇-X₁₈-Ala-X₂₀-X₂₁-Phe-Val-X₂₄-Trp-Leu-X₂₇-X₂₈-X₂₉-X₃₀;a substituent comprising a lipophilic moiety and at least threenegatively charged moieties; and a C-terminal amide; wherein, X₂represents Aib, Acb or Acpr; X₃ represents Gln or His; X₁₀ representsLeu, Ile or Val; X₁₂ represents Lys or Arg; X₁₅ represents Asp or Glu;X₁₆ represents Ser, Ala, Leu, Thr, Glu, Aib, Ile, Val or Lys; X₁₇represents Arg or Lys; X₁₈ represents Arg, Ala or Lys; X₂₀ representsGln, Arg, Glu, Aib or Lys; X₂₁ represents Asp, Glu, Ser, or Lys; X₂₄represents Gln, Ala, Arg, Glu, Aib or Lys; X₂₇ represents Met, Leu orVal; X₂₈ represents Asn, Ser, Thr, Gln, Ala, Gly, Glu or Lys; X₂₉represents Thr, Gly, Ser, Gln, Ala, Glu or Lys; and X₃₀ represents Lysor is absent; wherein Lys is present at one or more positions selectedfrom the group consisting of X₁₂, X₁₆, X₁₇, X₁₈, X₂₀, X₂₁, X₂₄, X₂₈,X₂₉, and X₃₀; wherein said substituent comprisesZ₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀-; wherein Z₁ is

n is 6-20, and the symbol * represents the attachment point to thenitrogen of a neighbouring group; wherein Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈,Z₉, and Z₁₀ individually are absent or an amino acid selected from thegroup consisting of Glu, γGlu, Gly, Ser, Ala, Thr, and Ado; wherein atleast two of residues Z₂, Z₃, Z₄, Z₅, Z₆, Z₇, Z₈, Z₉, and Z₁₀ arepresent; wherein Z₁-Z₂-Z₃-Z₄-Z₅-Z₆-Z₇-Z₈-Z₉-Z₁₀ together contains atleast three negative charges; wherein one of said negatively chargedmoieties is distal of said lipophilic moiety; and wherein saidsubstituent is attached to said peptide at the epsilon position of a Lysresidue at one amino acid position selected from the group consisting ofX₁₆, X₁₇, X₁₈, X₂₀, X₂₁, X₂₄, X₂₈, X₂₉, and X₃₀; wherein the disease orcondition is selected from the group consisting of hyperglycemia, type 2diabetes, impaired glucose tolerance, type 1 diabetes, obesity, andoverweight.
 20. The method according to claim 21, wherein thesubstituent is selected from the group consisting of:

wherein * indicates the point of attachment to said peptide at thenitrogen atom of the epsilon position of a Lys residue.
 21. The methodaccording to claim 19, wherein said glucagon derivative is selected fromthe group consisting of:N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys6,Arg20,Leu27,Ser28]-Glucagonamide

N^(ε21)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]-Glucagonamide

N^(ε24)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Lys24,Leu27,Ser28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

N^(α)-([Aib2,Leu10,Arg20,Leu27,Ser28]-Glucagonyl)-N{Epsilon}[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]Lysamide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu1010,Lys16,Lys17,Glu21,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys17,Ala18,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala18,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Arg20,Glu21,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu16,Lys20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Thr16,Arg124,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Glu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Lys17,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagon amide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Ala18,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Lys6,Arg20,Glu21,Ala24,Leu27]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Ala18,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]-acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Val27,Lys28,Gly29]-Glucagonamide

N^(ε16)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Lys16,Glu21,Leu27]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Ala24,Leu27,Lys28]-Glucagon amide

N^(ε16)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys16,Arg20,Glu21,Ala24,Leu27,Ser28]-Glucagon amide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,His3,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,His3,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,His3,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagon amide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys29]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu1010,Arg12,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagon amide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acpr2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Aib16,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Leu16,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]-[Aib2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Glu15,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Arg12,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Arg12,Ala16,Arg20,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Acb2,Leu10,Glu15,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys29]-Glucagonamide

N^(ε29)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Ser28,Lys29]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu5,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Ser21,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Ala16,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Leu16,Leu27,Lys28]-Glucagonamide

N^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4)-4-carboxy-4-[[(4)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Ala16,Leu27,Lys28]-Glucagonamide

andN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Val10,Arg12,Leu16,Leu27,Lys28]-Glucagonamide


22. The method according to claim 19, wherein the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-Glucagonamide


23. The method according to claim 19, wherein the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-Glucagonamide


24. The method according to claim 19, wherein the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]amino]butanoyl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide


25. The method according to claim 19, wherein the glucagon derivative isN^(ε28)-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]-3-hydroxypropanoyl]amino]butanoyl]amino]butanoyl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-Glucagonamide


26. The method according to claim 19, wherein the glucagon derivative isN^(ε28)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-Glucagonamide